Antibodies that Bind Activatable Antibodies and Methods of Use Thereof

ABSTRACT

The invention relates generally to antibodies and antigen-binding fragments thereof that bind activatable antibodies and/or conjugated activatable antibodies and methods of making and using these antibodies that bind activatable antibodies and/or conjugated activatable antibodies.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/914,489, filed Dec. 11, 2013, the contents of which are incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to antibodies and antigen-bindingfragments thereof that bind activatable antibodies and/or conjugatedactivatable antibodies and methods of making and using these antibodiesthat bind activatable antibodies and/or conjugated activatableantibodies.

BACKGROUND OF THE INVENTION

Modified antibody-based therapies such as masked antibodies andactivatable antibodies have proven effective treatments for a variety ofdiseases.

Accordingly, there exists a need for reagents to detect the level ofsuch modified antibody-based therapeutics and to detect whether themodified antibody-based therapeutic is in an activated or non-activatedstate.

SUMMARY OF THE INVENTION

The invention provides antibodies and antigen-binding fragments thereofthat bind one or more activatable antibodies that include an antibody orantigen-binding fragment thereof (AB) that specifically binds a targetcoupled to a masking moiety (MM), such that coupling of the MM reducesthe ability of the antibody or antigen-binding fragment thereof to bindthe target. In some embodiments, the MM is coupled to the AB via asequence that includes a substrate for a protease, for example, aprotease that is co-localized with the target at a treatment site in asubject. This substrate sequence is also referred to herein as acleavable moiety (CM) sequence, and the terms substrate and CM are usedinterchangeably throughout.

In some embodiments, antibodies and antigen-binding fragments thereof ofthe disclosure bind one or more activatable antibodies that include anantibody or antigen-binding fragment thereof (AB) that specificallybinds epidermal growth factor receptor (EGFR) coupled to a maskingmoiety (MM), such that coupling of the MM reduces the ability of theantibody or antigen-binding fragment thereof to bind EGFR. In someembodiments, the MM is coupled to the AB via a sequence that includes asubstrate for a protease, for example, a protease that is co-localizedwith EGFR at a treatment site in a subject.

In some embodiments, antibodies and antigen-binding fragments thereof ofthe disclosure bind one or more activatable antibodies that include anantibody or antigen-binding fragment thereof (AB) that specificallybinds a Jagged target, e.g., Jagged 1 and/or Jagged 2, coupled to amasking moiety (MM), such that coupling of the MM reduces the ability ofthe antibody or antigen-binding fragment thereof to bind the Jaggedtarget. In some embodiments, the MM is coupled to the AB via a sequencethat includes a substrate for a protease, for example, a protease thatis co-localized with the Jagged target at a treatment site in a subject.

In some embodiments, antibodies and antigen-binding fragments thereof ofthe disclosure bind one or more activatable antibodies that include anantibody or antigen-binding fragment thereof (AB) that specificallybinds interleukin 6 receptor (IL-6R) coupled to a masking moiety (MM),such that coupling of the MM reduces the ability of the antibody orantigen-binding fragment thereof to bind IL-6R. In some embodiments, theMM is coupled to the AB via a sequence that includes a substrate for aprotease, for example, a protease that is co-localized with IL-6R at atreatment site in a subject.

The invention provides antibodies that bind conjugated activatableantibodies that include an antibody or antigen-binding fragment thereof(AB) that specifically binds a target coupled to a masking moiety (MM),such that coupling of the MM reduces the ability of the antibody orantigen-binding fragment thereof to bind the target, and wherein theactivatable antibody is conjugated to one or more additional agents. Insome embodiments of the conjugated activatable antibodies, the MM iscoupled to the AB via a sequence that includes a substrate for aprotease, for example, a protease that is co-localized with the targetat a treatment site in a subject. In some embodiments, the agent is atherapeutic agent. In some embodiments, the agent is an antineoplasticagent. In some embodiments, the agent is a toxin or fragment thereof. Insome embodiments, the agent is an agent selected from the group listedin Table 4. In some embodiments, the activatable antibody also includesa detectable moiety. In some embodiments, the detectable moiety is adiagnostic agent. In some embodiments, the detectable moiety is aconjugatable detection reagent. In some embodiments, the agent isconjugated to the AB via a linker. In some embodiments, the linker is acleavable linker.

The invention provides antibodies that bind conjugated activatableantibodies that include an antibody or antigen-binding fragment thereof(AB) that specifically binds epidermal growth factor receptor (EGFR)coupled to a masking moiety (MM), such that coupling of the MM reducesthe ability of the antibody or antigen-binding fragment thereof to bindEGFR, and wherein the activatable antibody is conjugated to one or moreadditional agents. In some embodiments of the conjugated activatableantibodies, the MM is coupled to the AB via a sequence that includes asubstrate for a protease, for example, a protease that is co-localizedwith EGFR at a treatment site in a subject.

The invention provides antibodies that bind conjugated activatableantibodies that include an antibody or antigen-binding fragment thereof(AB) that specifically binds a Jagged target, e.g., Jagged 1 and/orJagged 2, coupled to a masking moiety (MM), such that coupling of the MMreduces the ability of the antibody or antigen-binding fragment thereofto bind the Jagged target, and wherein the activatable antibody isconjugated to one or more additional agents. In some embodiments of theconjugated activatable antibodies, the MM is coupled to the AB via asequence that includes a substrate for a protease, for example, aprotease that is co-localized with the Jagged target at a treatment sitein a subject.

The invention provides antibodies that bind conjugated activatableantibodies that include an antibody or antigen-binding fragment thereof(AB) that specifically binds interleukin 6 receptor (IL-6R) coupled to amasking moiety (MM), such that coupling of the MM reduces the ability ofthe antibody or antigen-binding fragment thereof to bind IL-6R, andwherein the activatable antibody is conjugated to one or more additionalagents. In some embodiments of the conjugated activatable antibodies,the MM is coupled to the AB via a sequence that includes a substrate fora protease, for example, a protease that is co-localized with IL-6R at atreatment site in a subject.

The antibodies and antigen-binding fragments of the disclosure that bindactivatable antibodies and/or conjugated activatable antibodies arecollectively referred to herein as anti-AA antibodies and anti-AAantibody fragments.

In some embodiments, the antibody or fragment thereof that binds theactivatable antibody is a monoclonal antibody, domain antibody, singlechain, Fab fragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a singledomain heavy chain antibody, and a single domain light chain antibody.In some embodiments, such an antibody or fragment thereof that binds anactivatable antibody is a rabbit, mouse, chimeric, humanized or fullyhuman monoclonal antibody.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody has anequilibrium dissociation constant of about 100 nM or less for binding tothe activatable antibody and/or conjugated activatable antibody.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises aheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 6, 10, 14, 76, and 105. In some embodiments, the antibodyor fragment thereof that binds an activatable antibody and/or conjugatedactivatable antibody comprises a light chain amino acid sequenceselected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93,and 108. In some embodiments, the antibody or fragment thereof thatbinds an activatable antibody and/or conjugated activatable antibodycomprises a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 6, 10, 14, 76, and 105, and a light chainamino acid sequence selected from the group consisting of SEQ ID NO: 4,8, 12, 16, 86, 93, and 108.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a heavy chain and a light chain selected from the groupconsisting of a heavy chain comprising the amino acid sequence of SEQ IDNO: 2 and a light chain comprising the amino acid sequence of SEQ ID NO:4; a heavy chain comprising the amino acid sequence of SEQ ID NO: 6 anda light chain comprising the amino acid sequence of SEQ ID NO: 8; aheavy chain comprising the amino acid sequence of SEQ ID NO: 10 and alight chain comprising the amino acid sequence of SEQ ID NO: 12; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 14 and alight chain comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises aheavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and105. In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises alight chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93, and 108. Insome embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises aheavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and105, and a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93,and 108.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a heavy chain and a light chain selected from the groupconsisting of a heavy chain comprising the amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 2 and a light chain comprising theamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4; aheavy chain comprising the amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 6 and a light chain comprising the aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; a heavychain comprising the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 10 and a light chain comprising the amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 12; and a heavy chaincomprising the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 14 and a light chain comprising the amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises avariable heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 42, 56, 60, 64, 77, and 106. In someembodiments, the antibody or fragment thereof that binds an activatableantibody and/or conjugated activatable antibody comprises a variablelight chain amino acid sequence selected from the group consisting ofSEQ ID NO: 44, 58, 62, 66, 87, 94, and 109. In some embodiments, theantibody or fragment thereof that binds an activatable antibody and/orconjugated activatable antibody comprises a variable heavy chain aminoacid sequence selected from the group consisting of SEQ ID NO: 42, 56,60, 64, 77, and 106, and a variable light chain amino acid sequenceselected from the group consisting of SEQ ID NO: 44, 58, 62, 66, 87, 94,and 109.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a variable heavy chain and a variable light chainselected from the group consisting of a variable heavy chain comprisingthe amino acid sequence of SEQ ID NO: 42 and a variable light chaincomprising the amino acid sequence of SEQ ID NO: 44; a variable heavychain comprising the amino acid sequence of SEQ ID NO: 56 and a variablelight chain comprising the amino acid sequence of SEQ ID NO: 58; avariable heavy chain comprising the amino acid sequence of SEQ ID NO: 60and a variable light chain comprising the amino acid sequence of SEQ IDNO: 62; and a variable heavy chain comprising the amino acid sequence ofSEQ ID NO: 64 and a variable light chain comprising the amino acidsequence of SEQ ID NO: 66.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a variable heavy chain and a variable light chainselected from the group consisting of a variable heavy chain comprisingthe amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42 and a variable light chain comprising the amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 44; a variable heavy chaincomprising the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 56 and a variable light chain comprising the amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 58; a variableheavy chain comprising the amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 60 and a variable light chain comprising theamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 62;and a variable heavy chain comprising the amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 64 and a variable light chaincomprising the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 66.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises avariable heavy chain amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 42, 56, 60, 64, 77, and106. In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises avariable light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 44, 58, 62, 66, 87, 94,and 109. In some embodiments, the antibody or fragment thereof thatbinds an activatable antibody and/or conjugated activatable antibodycomprises a variable heavy chain amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an aminoacid sequence selected from the group consisting of SEQ ID NO: 42, 56,60, 64, 77, and 106, and a variable light chain amino acid that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 44,58, 62, 66, 87, 94, and 109.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a variable heavy chain complementarity determining region1 (VH CDR1, also referred to herein as CDRH1) sequence, a variable heavychain complementarity determining region 2 (VH CDR2, also referred toherein as CDRH2) sequence, a variable heavy chain complementaritydetermining region 3 (VH CDR3, also referred to herein as CDRH3)sequence, a variable light chain complementarity determining region 1(VL CDR1, also referred to herein as CDRL1) sequence, a variable lightchain complementarity determining region 2 (VL CDR2, also referred toherein as CDRL2) sequence, and a variable light chain complementaritydetermining region 3 (VL CDR3, also referred to herein as CDRL3)sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes at least an amino acidsequence selected from the group consisting of SEQ ID NO: 67, 73, 78,88, 95, and 101; a VH CD2 sequence that includes at least an amino acidsequence selected from the group consisting of SEQ ID NO: 68, 74, 79,89, 96, and 102; a VH CDR3 sequence that includes at least an amino acidsequence selected from the group consisting of SEQ ID NO: 69, 80, 90,and 97; a VL CDR1 sequence that includes at least an amino acid sequenceselected from the group consisting of SEQ ID NO: 70, 81, and 98; a VLCDR2 sequence that includes at least an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 71, 82, and 99; and a VL CDR3sequence that includes at least an amino acid sequence selected from thegroup consisting of SEQ ID NO: 72, 83, 84, 91, 110, 100, and 103.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein at least one CDR sequence is selected from the groupconsisting of a VH CDR1 sequence that includes a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto an amino acid sequence selected from the group consisting of SEQ IDNO: 67, 73, 78, 88, 95, and 101; a VH CD2 sequence that includes asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 68, 74, 79, 89, 96, and 102; a VH CDR3 sequencethat includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 69, 80, 90, and 97; a VL CDR1sequence that includes a sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 70, 81, and 98; a VLCDR2 sequence that includes a sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 71, 82, and99; and a VL CDR3 sequence that includes a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 72,83, 84, 91, 110, 100, and 103.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises at least an amino acidsequence selected from the group consisting of SEQ ID NO: 67, 73, 78,88, 95, and 101; the VH CD2 sequence comprises at least an amino acidsequence selected from the group consisting of SEQ ID NO: 68, 74, 79,89, 96, and 102; the VH CDR3 sequence comprises at least an amino acidsequence selected from the group consisting of SEQ ID NO: 69, 80, 90,and 97; the VL CDR1 sequence comprises at least an amino acid sequenceselected from the group consisting of SEQ ID NO: 70, 81, and 98; the VLCDR2 sequence comprises at least an amino acid sequence selected fromthe group consisting of SEQ ID NO: 71, 82, and 99; and the VL CDR3sequence comprises at least an amino acid sequence selected from thegroup consisting of SEQ ID NO: 72, 83, 84, 91, 110, 100, and 103.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto an amino acid sequence selected from the group consisting of SEQ IDNO: 67, 73, 78, 88, 95, and 101; a VH CD2 sequence comprises a sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 68, 74, 79, 89, 96, and 102; the VH CDR3sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identical to an amino acid sequenceselected from the group consisting of SEQ ID NO: 69, 80, 90, and 97; theVL CDR1 sequence comprises a sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 70, 81, and98; the VL CDR2 sequence comprises a sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an aminoacid sequence selected from the group consisting of SEQ ID NO: 71, 82,and 99; and the VL CDR3 sequence comprises a sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 72,83, 84, 91, 110, 100, and 103.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises the amino acid sequenceNYAVMC (SEQ ID NO: 67), the VH CDR2 sequence comprises the amino acidsequence CIVLGDGGTTYYASWARG (SEQ ID NO: 68), the VH CDR3 sequencecomprises the amino acid sequence SFAASSPINYFNL (SEQ ID NO: 69), the VLCDR1 sequence comprises the amino acid sequence QASQRISTYLA (SEQ ID NO:70), the VL CDR2 sequence comprises the amino acid sequence KASTLAS (SEQID NO: 71), and the VL CDR3 sequence comprises the amino acid sequenceQSYYFGDGTTFA (SEQ ID NO: 72).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence NYAVMC (SEQ ID NO: 67), the VHCDR2 sequence comprises an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence CIVLGDGGTTYYASWARG (SEQ ID NO: 68), the VH CDR3sequence comprises an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence SFAASSPINYFNL (SEQ ID NO: 69), the VL CDR1 sequencecomprises an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence QASQRISTYLA (SEQ ID NO: 70), the VL CDR2 sequence comprises anamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to the amino acid sequence KASTLAS (SEQID NO: 71), and the VL CDR3 sequence comprises an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence QSYYFGDGTTFA (SEQ ID NO: 72).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises the amino acid sequenceRYGMA (SEQ ID NO: 78), the VH CDR2 sequence comprises the amino acidsequence AISSSGNEDYASWAIG (SEQ ID NO: 79), the VH CDR3 sequencecomprises the amino acid sequence GWLSNNAYM (SEQ ID NO: 80), the VL CDR1sequence comprises the amino acid sequence QASQSIYNKNQLS (SEQ ID NO:81), the VL CDR2 sequence comprises the amino acid sequence YASTLAS (SEQID NO: 82), and the VL CDR3 sequence comprises the amino acid sequenceLGDFSCSGVDCLV (SEQ ID NO: 83).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence RYGMA (SEQ ID NO: 78), the VHCDR2 sequence comprises an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence AISSSGNEDYASWAIG (SEQ ID NO: 79), the VH CDR3sequence comprises an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence GWLSNNAYM (SEQ ID NO: 80), the VL CDR1 sequence comprisesan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceQASQSIYNKNQLS (SEQ ID NO: 81), the VL CDR2 sequence comprises an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the amino acid sequence YASTLAS (SEQ IDNO: 82), and the VL CDR3 sequence comprises an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the amino acid sequence LGDFSCSGVDCLV (SEQ ID NO: 83).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises the amino acid sequenceHYGMA (SEQ ID NO: 88), the VH CDR2 sequence comprises the amino acidsequence AISSSGNEDYASWPKG (SEQ ID NO: 89), the VH CDR3 sequencecomprises the amino acid sequence GWLSNNVYM (SEQ ID NO: 90), the VL CDR1sequence comprises the amino acid sequence QASQSIYNKNQLS (SEQ ID NO:81), the VL CDR2 sequence comprises the amino acid sequence YASTLAS (SEQID NO: 82), and the VL CDR3 sequence comprises the amino acid sequenceLGDFSCSGVDCLS (SEQ ID NO: 91).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence HYGMA (SEQ ID NO: 88), the VHCDR2 sequence comprises an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence AISSSGNEDYASWPKG (SEQ ID NO: 89), the VH CDR3sequence comprises an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence GWLSNNVYM (SEQ ID NO: 90), the VL CDR1 sequence comprisesan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceQASQSIYNKNQLS (SEQ ID NO: 81), the VL CDR2 sequence comprises an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the amino acid sequence YASTLAS (SEQ IDNO: 82), and the VL CDR3 sequence comprises an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the amino acid sequence LGDFSCSGVDCLS (SEQ ID NO: 91).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises the amino acid sequenceSYCMS (SEQ ID NO: 95), the VH CDR2 sequence comprises the amino acidsequence IIGGICSTYYAAWAKG (SEQ ID NO: 96), the VH CDR3 sequencecomprises the amino acid sequence PAYNSDPI (SEQ ID NO: 97), the VL CDR1sequence comprises the amino acid sequence QASQSVYNNNYLS (SEQ ID NO:98), the VL CDR2 sequence comprises the amino acid sequence DAATLAS (SEQID NO: 99), and the VL CDR3 sequence comprises the amino acid sequenceLGEFSCGSADCNA (SEQ ID NO: 100).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody comprises acombination of a VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3sequence, a VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3sequence, wherein the VH CDR1 sequence comprises an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the amino acid sequence SYCMS (SEQ ID NO: 95), the VHCDR2 sequence comprises an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to theamino acid sequence IIGGICSTYYAAWAKG (SEQ ID NO: 96), the VH CDR3sequence comprises an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the aminoacid sequence PAYNSDPI (SEQ ID NO: 97), the VL CDR1 sequence comprisesan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the amino acid sequenceQASQSVYNNNYLS (SEQ ID NO: 98), the VL CDR2 sequence comprises an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the amino acid sequence DAATLAS (SEQ IDNO: 99), and the VL CDR3 sequence comprises an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the amino acid sequence LGEFSCGSADCNA (SEQ ID NO: 100).

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a heavy chain amino acid sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14,76, and 105. In some embodiments, the antibody or fragment thereof thatbinds an activatable antibody and/or conjugated activatable antibody isencoded by a nucleic acid sequence that comprises a nucleic acidsequence that comprises a nucleic acid sequence encoding a light chainamino acid sequence comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93, and 108. In someembodiments, the antibody or fragment thereof that binds an activatableantibody and/or conjugated activatable antibody is encoded by a nucleicacid sequence that comprises a nucleic acid sequence encoding a heavychain amino acid sequence comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and 105, and anucleic acid sequence that comprises a nucleic acid sequence encoding alight chain amino acid sequence comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93,and 108.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 6, 10, 14,76, and 105. In some embodiments, the antibody or fragment thereof thatbinds an activatable antibody and/or conjugated activatable antibody isencoded by a nucleic acid sequence that comprises a nucleic acidsequence that comprises a nucleic acid sequence encoding a light chainamino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 8, 12, 16, 86, 93, and 108. In someembodiments, the antibody or fragment thereof that binds an activatableantibody and/or conjugated activatable antibody is encoded by a nucleicacid sequence that comprises a nucleic acid sequence encoding a heavychain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and 105, and anucleic acid sequence that comprises a nucleic acid sequence encoding alight chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO: 4, 8, 12, 16, 86, 93, and 108.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a combination of a heavy chain and a light chain selected fromthe group consisting of a heavy chain and a light chain selected fromthe group consisting of a heavy chain comprising the amino acid sequenceof SEQ ID NO: 2 and a light chain comprising the amino acid sequence ofSEQ ID NO: 4; a heavy chain comprising the amino acid sequence of SEQ IDNO: 6 and a light chain comprising the amino acid sequence of SEQ ID NO:8; a heavy chain comprising the amino acid sequence of SEQ ID NO: 10 anda light chain comprising the amino acid sequence of SEQ ID NO: 12; and aheavy chain comprising the amino acid sequence of SEQ ID NO: 14 and alight chain comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a combination of a heavy chain and a light chain selected fromthe group consisting of a heavy chain comprising the amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence of SEQ ID NO: 2 and a light chaincomprising the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 4; a heavy chain comprising the amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 6 and a light chain comprising theamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; aheavy chain comprising the amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 10 and a light chain comprising the aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 12; and aheavy chain comprising the amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO: 14 and a light chain comprising the aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a variable heavy chain amino acid sequence comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 42, 56,60, 64, 77, and 106. In some embodiments, the antibody or fragmentthereof that binds an activatable antibody and/or conjugated activatableantibody is encoded by a nucleic acid sequence that comprises a nucleicacid sequence encoding a variable light chain amino acid sequencecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 44, 58, 62, 66, 87, 94, and 109. In some embodiments, theantibody or fragment thereof that binds an activatable antibody and/orconjugated activatable antibody is encoded by a nucleic acid sequencethat comprises a nucleic acid sequence encoding a variable heavy chainamino acid sequence comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 42, 56, 60, 64, 77, and 106, and anucleic acid sequence that comprises a nucleic acid sequence encoding avariable light chain amino acid sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 44, 58, 62,66, 87, 94, and 109.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 42, 56, 60,64, 77, and 106. In some embodiments, the antibody or fragment thereofthat binds an activatable antibody and/or conjugated activatableantibody is encoded by a nucleic acid sequence that comprises a nucleicacid sequence encoding a light chain amino acid that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 44, 58, 62,66, 87, 94, and 109. In some embodiments, the antibody or fragmentthereof that binds an activatable antibody and/or conjugated activatableantibody is encoded by a nucleic acid sequence that comprises a nucleicacid sequence encoding a heavy chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence selected from the group consisting of SEQ ID NO: 42,56, 60, 64, 77, and 106, and a nucleic acid sequence that comprises anucleic acid sequence encoding a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an aminoacid sequence selected from the group consisting of SEQ ID NO: 44, 58,62, 66, 87, 94, and 109.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a combination of a variable heavy chain and a variable lightchain selected from the group consisting of a variable heavy chaincomprising the amino acid sequence of SEQ ID NO: 42 and a variable lightchain comprising the amino acid sequence of SEQ ID NO: 44; a variableheavy chain comprising the amino acid sequence of SEQ ID NO: 56 and avariable light chain comprising the amino acid sequence of SEQ ID NO:58; a variable heavy chain comprising the amino acid sequence of SEQ IDNO: 60 and a variable light chain comprising the amino acid sequence ofSEQ ID NO: 62; and a variable heavy chain comprising the amino acidsequence of SEQ ID NO: 64 and a variable light chain comprising theamino acid sequence of SEQ ID NO: 66.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a nucleic acid sequenceencoding a combination of a variable heavy chain and a variable lightchain selected from the group consisting of a variable heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence of SEQ ID NO: 42 and a variable light chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 44;a variable heavy chain comprising an amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto the amino acid sequence of SEQ ID NO: 56 and a variable light chaincomprising the amino acid sequence of SEQ ID NO: 58; a variable heavychain comprising an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence of SEQ ID NO: 60 and a variable light chain an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to the amino acid sequence of SEQ ID NO: 62; and avariable heavy chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical tothe amino acid sequence of SEQ ID NO: 64 and a variable light chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence of SEQ ID NO: 66.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a heavy chain nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, 5, 9, and 13. In some embodiments, theantibody or fragment thereof that binds an activatable antibody and/orconjugated activatable antibody is encoded by a light chain nucleic acidsequence selected from the group consisting of SEQ ID NO: 3, 7, 11, and15. In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a heavy chain nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, 5, 9, and 13, and a light chain nucleic acidsequence selected from the group consisting of SEQ ID NO: 3, 7, 11, and15.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a heavy chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, 5, 9, and 13. In some embodiments, theantibody or fragment thereof that binds an activatable antibody and/orconjugated activatable antibody is encoded by a light chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a light chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 3, 7, 11, and 15. In some embodiments,the antibody or fragment thereof that binds an activatable antibodyand/or conjugated activatable antibody is encoded by a heavy chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to a heavy chain nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1, 5, 9, and 13, and alight chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a light chain nucleic acidsequence selected from the group consisting of SEQ ID NO: 3, 7, 11, and15.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid encoding a heavy chain and a nucleic acid encoding alight chain selected from the group consisting of a heavy chain nucleicacid sequence comprising the nucleic acid sequence of SEQ ID NO: 1 and alight chain nucleic acid sequence comprising the nucleic acid sequenceof SEQ ID NO: 3; a heavy chain nucleic acid sequence comprising thenucleic acid sequence of SEQ ID NO: 5 and a light chain nucleic acidsequence comprising the nucleic acid sequence of SEQ ID NO: 7; a heavychain nucleic acid sequence comprising the nucleic acid sequence of SEQID NO: 9 and a light chain nucleic acid sequence comprising the nucleicacid sequence of SEQ ID NO: 11; and a heavy chain nucleic acid sequencecomprising the nucleic acid sequence of SEQ ID NO: 13 and a light chainnucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:15.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid encoding a heavy chain and a nucleic acid encoding alight chain selected from the group consisting of a heavy chain nucleicacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 1 and alight chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence ofSEQ ID NO: 3; a heavy chain nucleic acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequence of SEQ ID NO: 5 and a light chain nucleic acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequence of SEQ ID NO: 7; a heavy chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ IDNO: 9 and a light chain nucleic acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence of SEQ ID NO: 11; and a heavy chain nucleic acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the nucleic acid sequence of SEQ ID NO: 13 and a light chain nucleicacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the nucleic acid sequence of SEQ ID NO: 15.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a heavy chain nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 41, 55, 59, and 63. In some embodiments, theantibody or fragment thereof that binds an activatable antibody and/orconjugated activatable antibody is encoded by a light chain nucleic acidsequence selected from the group consisting of SEQ ID NO: 43, 57, 61,and 65. In some embodiments, the antibody or fragment thereof that bindsan activatable antibody and/or conjugated activatable antibody isencoded by a heavy chain nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 41, 55, 59, and 63, and a light chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 43, 57,61, and 65.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence that comprises a heavy chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 41, 55, 59, and 63. In some embodiments,the antibody or fragment thereof that binds an activatable antibodyand/or conjugated activatable antibody is encoded by a light chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to a light chain nucleic acid sequenceselected from the group consisting of SEQ ID NO: 43, 57, 61, and 65. Insome embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a heavy chain nucleic acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 41, 55,59, and 63, and a light chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a lightchain nucleic acid sequence selected from the group consisting of SEQ IDNO: 43, 57, 61, and 65.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid encoding a variable heavy chain and a nucleic acidencoding a variable light chain selected from the group consisting of avariable heavy chain nucleic acid sequence comprising the nucleic acidsequence of SEQ ID NO: 41 and a variable light chain nucleic acidsequence comprising the nucleic acid sequence of SEQ ID NO: 43; avariable heavy chain nucleic acid sequence comprising the nucleic acidsequence of SEQ ID NO: 55 and a variable light chain nucleic acidsequence comprising the nucleic acid sequence of SEQ ID NO: 57; avariable heavy chain nucleic acid sequence comprising the nucleic acidsequence of SEQ ID NO: 59 and a variable light chain nucleic acidsequence comprising the nucleic acid sequence of SEQ ID NO: 61; and avariable heavy chain nucleic acid sequence comprising the nucleic acidsequence of SEQ ID NO: 63 and a variable light chain nucleic acidsequence comprising the nucleic acid sequence of SEQ ID NO: 65.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid encoding a variable heavy chain and a nucleic acidencoding a variable light chain selected from the group consisting of avariable heavy chain nucleic acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence of SEQ ID NO: 41 and a variable light chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the nucleic acid sequence of SEQ ID NO: 43; a variableheavy chain nucleic acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence ofSEQ ID NO: 55 and a variable light chain nucleic acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe nucleic acid sequence of SEQ ID NO: 57; a variable heavy chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ IDNO: 59 and a variable light chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequence of SEQ ID NO: 61; and a variable heavy chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ IDNO: 63 and a variable light chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequence of SEQ ID NO: 65.

In some embodiments, the nucleic acid encoding the isolated antibodycomprises a nucleic acid encoding a signal peptide.

In some embodiments, the nucleic acid encoding the signal peptide isoperably linked to the nucleic acid encoding the activatable antibodyvia a nucleic acid encoding a spacer.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody is anantibody or is derived from an antibody selected from the groupconsisting of 10 (also referred to herein as 10-10 and/or clone 10), 8(also referred to herein as 8-8 and/or clone 8), 53 (also referred toherein as 53-1 and/or clone 3), 7 (also referred to herein as 7-11and/or clone 7), 36 (also referred to herein as 36-3 and/or clone 36),52 (also referred to herein as 52-10 and/or clone 52), and 27 (alsoreferred to herein as 27-4 and/or clone 27), and antigen-bindingfragments thereof.

The disclosure also provides vector(s) that include one or more of anucleic acid encoding an antibody or fragment thereof that binds anactivatable antibody. The disclosure also provides methods producing anisolated antibody or fragment thereof that binds an activatable antibodyby culturing a cell under conditions that lead to expression of theisolated antibody, wherein the cell includes a vector of the disclosure.

In some embodiments, the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody alsoincludes a detectable moiety. In some embodiments, the detectable moietyis a diagnostic agent. In some embodiments, the detectable moiety is aconjugatable detection reagent.

In some embodiments, the detectable moiety includes an imaging agent, acontrasting agent, an enzyme, a fluorescent label, a chromophore, a dye,one or more metal ions, or a ligand-based label. In some embodiments,the imaging agent comprises a radioisotope. In some embodiments, theradioisotope is indium or technetium. In some embodiments, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments, the enzyme comprises horseradish peroxidase, alkalinephosphatase, or β-galactosidase. In some embodiments, the fluorescentlabel comprises yellow fluorescent protein (YFP), cyan fluorescentprotein (CFP), green fluorescent protein (GFP), modified red fluorescentprotein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, ora europium derivative. In some embodiments, the detectable moiety is,for example, a fluorescein derivative such as fluorescein isothiocyanate(FITC). In some embodiments, the luminescent label comprises anN-methylacrydium derivative. In some embodiments, the label comprises anAlexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In someembodiments, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

The invention provides methods of detecting the presence of activatableantibody and/or conjugated activatable antibody in a sample or a subjectusing one or more of the antibody or fragment thereof that binds anactivatable antibody and/or conjugated activatable antibody describedherein.

Compositions according to the invention can include an antibody orfragment thereof that binds an activatable antibody and/or conjugatedactivatable antibody and a carrier. These compositions can be includedin kits, such as, for example, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a series of graphs depicting the binding specificity ofvarious antibodies of the disclosure for the activatable anti-EGFRantibody referred to herein as 3954-1204-c225v5.

FIGS. 2A and 2B are a series of graphs depicting the results of an ELISAthat uses antibodies and antigen-binding fragments thereof of thedisclosure that bind activatable antibodies and/or conjugatedactivatable antibodies to measure the concentration of total (activatedand non-activated) activatable anti-EGFR antibody or intact(non-activated) activatable anti-EGFR antibody.

FIG. 3 is a photograph depicting the staining by antibody 41-2 (alsoreferred to herein as antibody 41) observed in H292 and LXFA677 tumorsexcised from mice treated with (i) anti-EGFR antibody cetuximab (col.1), (ii) the activatable anti-EGFR antibody 3954-1204-c225v5 (col. 2),(iii) the masked anti-EGFR antibody 3954-NSUB-c225v5 which contains anon-cleavable sequence in lieu of the substrate sequence in3954-1204-c225v5 (col. 3), or (iv) PBS (col. 4).

FIG. 4 is a graph depicting the effect of human serum on the binding ofantibodies to the anti-Jagged 1/Jagged 2 activatable antibody referredto herein as 5342-1204-4D11 (anti-Jagged AA antibodies).

DETAILED DESCRIPTION OF THE INVENTION

Antibodies and antigen-binding fragments thereof described hereinspecifically bind an activatable antibody and/or conjugated activatableantibody. Also included in the disclosure are antibodies andantigen-binding fragments thereof that bind to the same epitope as theantibodies and antigen-binding fragments thereof that bind toactivatable antibodies and/or conjugated activatable antibodies.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., a rabbit monoclonal, a mouse monoclonal or humanized antibody)has the same specificity as a monoclonal antibody used in the methodsdescribed herein by ascertaining whether the former prevents the latterfrom binding to an activatable antibody and/or conjugated activatableantibody. If the monoclonal antibody being tested competes with themonoclonal antibody of the invention, as shown by a decrease in bindingby the monoclonal antibody of the invention, then the two monoclonalantibodies bind to the same, or a closely related, epitope. Analternative method for determining whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with an activatable antibodyand/or conjugated activatable antibody and then add the monoclonalantibody being tested to determine if the monoclonal antibody beingtested is inhibited in its ability to bind the activatable antibodyand/or conjugated activatable antibody. If the monoclonal antibody beingtested is inhibited then, in all likelihood, it has the same, orfunctionally equivalent, epitopic specificity as the monoclonal antibodyof the invention.

Exemplary antibodies that bind activatable antibodies and/or conjugatedactivatable antibodies include the antibodies referred to herein as 41(also referred to as 41-2 and/or clone 41), 58 (also referred to hereinas 58-1 and/or clone 58), 72 (also referred to herein as 72-3 and/orclone 72), 85 (also referred to herein as 85-1 and/or clone 85), 10(also referred to herein as 10-10 and/or clone 10), 8 (also referred toherein as 8-8 and/or clone 8), 53 (also referred to herein as 53-1and/or clone 3), 7 (also referred to herein as 7-11 and/or clone 7), 36(also referred to herein as 36-3 and/or clone 36), 52 (also referred toherein as 52-10 and/or clone 52), and 27 (also referred to herein as27-4 and/or clone 27).

Antibody 41-2 has the amino acid and nucleic acid sequences shown below.The heavy chain complementarity determining regions (CDRs) and lightchain CDRs are shown in boxes in the amino acid sequences presentedbelow. In particular, the antibody referred to herein as 41-2 includes aheavy chain CDR1 (CDRH1) sequence that comprises the amino acid sequenceNYAVMC (SEQ ID NO: 67), a heavy chain CDR2 (CDRH2) sequence thatcomprises the amino acid sequence CIVLGDGGTTYYASWARG (SEQ ID NO: 68), aheavy chain CDR3 (CDRH3) sequence that comprises the amino acid sequenceSFAASSPINYFNL (SEQ ID NO: 69), a light chain CDR1 (CDRL1) sequence thatcomprises the amino acid sequence QASQRISTYLA (SEQ ID NO: 70), a lightchain CDR2 (CDRL2) sequence that comprises the amino acid sequenceKASTLAS (SEQ ID NO: 71), and a light chain CDR3 (CDRL3) sequence thatcomprises the amino acid sequence QSYYFGDGTTFA (SEQ ID NO: 72). The CDRsof the anti-activatable antibodies of the disclosure are identifiedaccording to Kabat, E. A., et al. (1991) Sequences of Protein ofImmunological Interest, 5th edition, Public Health Service, NationalInstitutes of Health, Bethesda, Md.

41-2 Heavy Chain (H3) Nucleic Acid Sequence with 5′Sequence Including HindIII RestrictionSite (underlined), Signal Peptide (bold), and 3′Sequence Including Sequence Including NotIRestriction Site (underlined): (SEQ ID NO: 17) AAGCTTGTACCCTTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGCAGGAGTCCGGGGGAGGCCTGTTCCAGCCTGGGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAATTATGCCGTGATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTCTTGGTGATGGTGGTACTACTTATTACGCGAGCTGGGCGAGAGGCCGGTTCACCATCTCCAAACCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACGGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAGTTTTGCTGCTAGTAGCCCCATTAACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGAGCTGCGGCCGC41-2 Heavy Chain (H3) Nucleic Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 18)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGCAGGAGTCCGGGGGAGGCCTGTTCCAGCCTGGGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAATTATGCCGTGATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTCTTGGTGATGGTGGTACTACTTATTACGCGAGCTGGGCGAGAGGCCGGTTCACCATCTCCAAACCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACGGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAGTTTTGCTGCTAGTAGCCCCATTAACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 41-2 Heavy Chain (H3) Nucleic Acid Sequence: (SEQ ID NO: 1)CAGTCGCTGCAGGAGTCCGGGGGAGGCCTGTTCCAGCCTGGGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAATTATGCCGTGATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTCTTGGTGATGGTGGTACTACTTATTACGCGAGCTGGGCGAGAGGCCGGTTCACCATCTCCAAACCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACGGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAGTTTTGCTGCTAGTAGCCCCATTAACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 41-2 Heavy Chain Variable Region Nucleic Acid Sequence: (SEQ ID NO: 41)CAGTCGCTGCAGGAGTCCGGGGGAGGCCTGTTCCAGCCTGGGGGATCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCCTCAGTAATTATGCCGTGATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGTATTGTTCTTGGTGATGGTGGTACTACTTATTACGCGAGCTGGGCGAGAGGCCGGTTCACCATCTCCAAACCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACGGCCGCGGACACGGCCACCTATTTCTGTGCGAGAAGTTTTGCTGCTAGTAGCCCCATTAACTACTTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA 41-2 Heavy Chain (H3) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 19)

TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  41-2 Heavy Chain (H3) Amino Acid Sequence:(SEQ ID NO: 2)

SVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*41-2 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 42)

41-2 Light Chain (L2) Nucleic Acid Sequence with 5′Sequence Including HindIII RestrictionSite (underlined), Signal Peptide (bold), and 3′Sequence Including NotI Restriction Site (underlined): (SEQ ID NO: 20)AAGCTTGTACCCTTCACC ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTTCATCCCCCGTGTCTGCACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGCGCATTAGTACCTACCTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGCATCTGGGACAGAGTTCACTCTCACCATCAACGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAGAGCTATTATTTTGGTGATGGTACTACTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGCGAGAGCGGCCGC41-2 Light Chain (L2) Nucleic Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 21)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTTCATCCCCCGTGTCTGCACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGCGCATTAGTACCTACCTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGCATCTGGGACAGAGTTCACTCTCACCATCAACGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAGAGCTATTATTTTGGTGATGGTACTACTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG  41-2 Light Chain (L2) Nucleic Acid Sequence: (SEQ ID NO: 3)CAAGTGCTGACCCAGACTTCATCCCCCGTGTCTGCACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGCGCATTAGTACCTACCTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGCATCTGGGACAGAGTTCACTCTCACCATCAACGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAGAGCTATTATTTTGGTGATGGTACTACTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTT AG 41-2 Light Chain Variable Region Nucleic Acid Sequence: (SEQ ID NO: 43)CAAGTGCTGACCCAGACTTCATCCCCCGTGTCTGCACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGCGCATTAGTACCTACCTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGCATCTGGGACAGAGTTCACTCTCACCATCAACGACCTGGAGTGTGACGATGCTGCCACTTACTACTGTCAGAGCTATTATTTTGGTGATGGTACTACTTTTGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA 41-2 Light Chain (L2) Amino Acid Sequence with Signal Peptide (bold)(SEQ ID NO: 22)

VVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* 41-2 Light Chain (L2) Amino Acid Sequence: (SEQ ID NO: 4)

TGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* 41-2 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 44)

In some embodiments, antibody 41-2 is modified to remove one or morecysteine residues in one or more CDR sequences. In some embodiments, theantibody includes a CDRH1 sequence that comprises the amino acidsequence NYAVMX (SEQ ID NO: 73), where X is Ala or Ser, a CDRH2 sequencethat comprises the amino acid sequence XIVLGDGGTTYYASWARG (SEQ ID NO:74), where X is Ala or Ser, a CDRH3 sequence that comprises the aminoacid sequence SFAASSPINYFNL (SEQ ID NO: 69), a CDRL1 sequence thatcomprises the amino acid sequence QASQRISTYLA (SEQ ID NO: 70), a CDRL2sequence that comprises the amino acid sequence KASTLAS (SEQ ID NO: 71),and a CDRL3 sequence that comprises the amino acid sequence QSYYFGDGTTFA(SEQ ID NO: 72). In some embodiments, the antibody includes thefollowing heavy chain amino acid sequences:

41-2 Heavy Chain (H3) Amino Acid Sequence with Signal Peptide  (bold):(SEQ ID NO: 75)

TFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  Where X is Ala or Ser41-2 Heavy Chain (H3) Amino Acid Sequence: (SEQ ID NO: 76)

SVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFICSVMHEALHNHYTQKSISRSPGK*Where X is Ala or Ser41-2 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 77)

Where X is Ala or Ser

Antibody 58-1 has the amino acid and nucleic acid sequences shown below.The heavy chain complementarity determining regions (CDRs) and lightchain CDRs are shown in boxes in the amino acid sequences presentedbelow. In particular, the antibody referred to herein as 58-1 includes aCDRH1 sequence that comprises the amino acid sequence RYGMA (SEQ ID NO:78), a CDRH2 sequence that comprises the amino acid sequenceAISSSGNEDYASWAIG (SEQ ID NO: 79), a CDRH3 sequence that comprises theamino acid sequence GWLSNNAYM (SEQ ID NO: 80), a CDRL1 sequence thatcomprises the amino acid sequence QASQSIYNKNQLS (SEQ ID NO: 81), a CDRL2sequence that comprises the amino acid sequence YASTLAS (SEQ ID NO: 82),and a CDRL3 sequence that comprises the amino acid sequenceLGDFSCSGVDCLV (SEQ ID NO: 83).

58-1 Heavy Chain (H2) Nucleic Acid Sequence with 5′ Sequence IncludingHindIII Restriction Site (underlined), Signal Peptide (bold), and 3′Sequence Including NotI Restriction Site (underlined): (SEQ ID NO: 23)AAGCTTGTACCCTTCACC ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGTCGCTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGAAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGGCGATAGGCCGATTTACCATCTCCAAAACCTCGACCACGGCGGAGCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAACGCTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGAGCTGCGGCCGC 58-1 Heavy Chain (H2) Nucleic Acid Sequence with Signal Peptide  (bold):(SEQ ID NO: 24)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGTCGCTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGAAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGGCGATAGGCCGATTTACCATCTCCAAAACCTCGACCACGGCGGAGCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAACGCTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 58-1 Heavy Chain (H2) Nucleic Acid Sequence: (SEQ ID NO: 5)CAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGTCGCTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGAAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGGCGATAGGCCGATTTACCATCTCCAAAACCTCGACCACGGCGGAGCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAACGCTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 58-1 Heavy Chain Variable Region Nucleic Acid Sequence: (SEQ ID NO: 55)CAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGTACAGTCTCTGGAATCGACCTCAGTCGCTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGAAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGGCGATAGGCCGATTTACCATCTCCAAAACCTCGACCACGGCGGAGCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAACGCTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCA 58-1 Heavy Chain (H2) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 25)

VTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  58-1 Heavy Chain (H2) Amino Acid Sequence: (SEQ ID NO: 6)

CGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* 58-1 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 56)

58-1 Light Chain (L2) Nucleic Acid Sequence with 5′ Sequence   Including HindIll Restriction Site (underlined), Signal  Peptide (bold),and 3′ Sequence Including NotI Restriction Site (underlined): (SEQ ID NO: 26) AAGCTTGTACCCTTCACCATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCCGGTGCTGACCCAGACTCCAACGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTGTTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAATACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGCGAGAGCGGCCGC 58-1 Light Chain (L2) Nucleic Acid Sequence Signal Peptide (bold):(SEQ ID NO: 27)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCCGGTGCTGACCCAGACTCCAACGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTGTTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAATACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG  58-1 Light Chain (L2) Nucleic Acid Sequence:(SEQ ID NO: 7)CCGGTGCTGACCCAGACTCCAACGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTGTTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAATACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG  58-1 Light Chain Variable Region Nucleic Acid Sequence:(SEQ ID NO: 57)CCGGTGCTGACCCAGACTCCAACGCCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTGTTGTCGGCGGAGGGACCGAGGTGGTCGTCGAA 58-1 Light Chain (L2) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 28)

GTEVVVEGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*58-1 Light Chain (L2) Amino Acid Sequence: (SEQ ID NO: 8)

LVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*58-1 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 58)

In some embodiments, antibody 58-1 is modified to remove one or morecysteine residues in one or more CDR sequences. In some embodiments, theantibody includes a CDRH1 sequence that comprises the amino acidsequence RYGMA (SEQ ID NO: 78), a CDRH2 sequence that comprises theamino acid sequence AISSSGNEDYASWAIG (SEQ ID NO: 79), a CDRH3 sequencethat comprises the amino acid sequence GWLSNNAYM (SEQ ID NO: 80), aCDRL1 sequence that comprises the amino acid sequence QASQSIYNKNQLS (SEQID NO: 81), a CDRL2 sequence that comprises the amino acid sequenceYASTLAS (SEQ ID NO: 82), and a CDRL3 sequence that comprises the aminoacid sequence LGDFSXSGVDXLV (SEQ ID NO: 84), where X is Ala or Ser. Insome embodiments, the antibody includes the following light chain aminoacid sequences:

58-1 Light Chain (L2) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 85)

GTEVVVEGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser58-1 Light Chain (L2) Amino Acid Sequence: (SEQ ID NO: 86)

LVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser58-1 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 87)

Where X is Ala or Ser

Antibody 72-3 has the amino acid and nucleic acid sequences shown below.The heavy chain complementarity determining regions (CDRs) and lightchain CDRs are shown in boxes in the amino acid sequences presentedbelow. In particular, the antibody referred to herein as 72-3 includes aCDRH1 sequence that comprises the amino acid sequence HYGMA (SEQ ID NO:88), a CDRH2 sequence that comprises the amino acid sequenceAISSSGNEDYASWPKG (SEQ ID NO: 89), a CDRH3 sequence that comprises theamino acid sequence GWLSNNVYM (SEQ ID NO: 90), a CDRL1 sequence thatcomprises the amino acid sequence QASQSIYNKNQLS (SEQ ID NO: 81), a CDRL2sequence that comprises the amino acid sequence YASTLAS (SEQ ID NO: 82),and a CDRL3 sequence that comprises the amino acid sequenceLGDFSCSGVDCLS (SEQ ID NO: 91).

72-3 Heavy Chain (H1) Nucleic Acid Sequence with 5′ Sequence   Including HindIII Restriction Site (underlined), Signal Peptide    (bold), and 3′  Sequence Including NotI Restriction Site  (underlined):(SEQ ID NO: 29) AAGCTTGTACCCTTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCACTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGCCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGACTCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAATGTTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGAGCTG CGGCCGC72-3 Heavy Chain (H1) Nucleic Acid Sequence with Signal Peptide  (bold):(SEQ ID NO: 30)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCACTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGCCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGACTCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAATGTTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 72-3 Heavy Chain (H1) Nucleic Acid Sequence: (SEQ ID NO: 9)CAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCACTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGCCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGACTCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAATGTTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 72-3 Heavy Chain Variable Region Nucleic Acid Sequence: (SEQ ID NO: 59)CAGTCGGTGGAGGAGTCCGGGGGTCGTCTGGTCATGCCTGGAGGATCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCACTATGGAATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAATACATCGGAGCCATTAGTAGTAGTGGTAATGAAGACTACGCGAGCTGGCCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGACTCTGAAAATGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAGGTTGGCTTAGTAATAATGTTTATATGTGGGGCCCAGGCACCCTGGTCACCGTCTCGTCA 72-3 Heavy Chain (H1) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 31)

VTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  72-3 Heavy Chain (H1) Amino Acid Sequence: (SEQ ID NO: 10)

CGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* 72-3 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 60)

72-3 Light Chain (L1) Nucleic Acid Sequence with 5′ Sequence  Including HindIII Restriction Site (underlined), Signal Peptide   (bold), and 3′ Sequence Including NotI Restriction Site  (underlined):(SEQ ID NO: 32) AAGCTTGTACCCTTCACCATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGCTTCAGCAGAAACCAGGGCAGCCTCCCAAGGTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTTCTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGCGAGAGCGGCCGC 72-3 Light Chain (L1) Nucleic Acid Sequence with Signal Peptide  (bold):(SEQ ID NO: 33)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGCTTCAGCAGAAACCAGGGCAGCCTCCCAAGGTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTTCTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG  72-3 Light Chain (L1) Nucleic Acid Sequence:(SEQ ID NO: 11)CAAGTGCTGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGCTTCAGCAGAAACCAGGGCAGCCTCCCAAGGTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTTCTGTCGGCGGAGGGACCGAGGTGGTCGTCGAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCATCTGCTGATCTTGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG  72-3 Light Chain Variable Region Nucleic Acid Sequence:(SEQ ID NO: 61)CAAGTGCTGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAAAGTATTTATAATAAAAATCAATTATCCTGGCTTCAGCAGAAACCAGGGCAGCCTCCCAAGGTCCTGATCCATTATGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGATTTTAGTTGTAGTGGTGTTGATTGTCTTTCTGTCGGCGGAGGGACCGAGGTGGTCGTCGAA 72-3 Light Chain (L1) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 34)

GTEVVVEGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* 72-3 Light Chain (L1) Amino Acid Sequence: (SEQ ID NO: 12)

LVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*72-3 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 62)

In some embodiments, antibody 72-3 is modified to remove one or morecysteine residues in one or more CDR sequences. In some embodiments, theantibody includes a CDRH1 sequence that comprises the amino acidsequence HYGMA (SEQ ID NO: 88), a CDRH2 sequence that comprises theamino acid sequence AISSSGNEDYASWPKG (SEQ ID NO: 89), a CDRH3 sequencethat comprises the amino acid sequence GWLSNNVYM (SEQ ID NO: 90), aCDRL1 sequence that comprises the amino acid sequence QASQSIYNKNQLS (SEQID NO: 81), a CDRL2 sequence that comprises the amino acid sequenceYASTLAS (SEQ ID NO: 82), and a CDRL3 sequence that comprises the aminoacid sequence LGDFSXSGVDXLS (SEQ ID NO: 110), where X is Ala or Ser. Insome embodiments, the antibody includes the following light chain aminoacid sequences:

72-3 Light Chain (L1) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 92)

GTEVVVEGDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser72-3 Light Chain (L1) Amino Acid Sequence: (SEQ ID NO: 93)

LVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser72-3 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 94)

Where X is Ala or Ser

Antibody 85-1 has the amino acid and nucleic acid sequences shown below.The heavy chain complementarity determining regions (CDRs) and lightchain CDRs are shown in boxes in the amino acid sequences presentedbelow. In particular, the antibody referred to herein as 85-1 includes aCDRH1 sequence that comprises the amino acid sequence SYCMS (SEQ ID NO:95), a CDRH2 sequence that comprises the amino acid sequenceIIGGICSTYYAAWAKG (SEQ ID NO: 96), a CDRH3 sequence that comprises theamino acid sequence PAYNSDPI (SEQ ID NO: 97), a CDRL1 sequence thatcomprises the amino acid sequence QASQSVYNNNYLS (SEQ ID NO: 98), a CDRL2sequence that comprises the amino acid sequence DAATLAS (SEQ ID NO: 99),and a CDRL3 sequence that comprises the amino acid sequenceLGEFSCGSADCNA (SEQ ID NO: 100).

85-1 Heavy Chain (H1) Nucleic Acid Sequence with 5′ Sequence Including HindIII Restriction Site (underlined), Signal Peptide (bold), and    3′Sequence Including NotI Restriction Site (underlined): (SEQ ID NO: 35)AAGCTTGTACCCTTCACC ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTAGCTACTGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTGGTATCTGTAGCACATACTACGCAGCCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGACCTGCTTATAATAGTGACCCAATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGAGCTGCGG CCGC 85-1 Heavy Chain (H1) Nucleic Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 36)ATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTCAGTCGCTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTAGCTACTGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTGGTATCTGTAGCACATACTACGCAGCCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGACCTGCTTATAATAGTGACCCAATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA  85-1 Heavy Chain (H1) Nucleic Acid Sequence:(SEQ ID NO: 13)CAGTCGCTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTAGCTACTGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTGGTATCTGTAGCACATACTACGCAGCCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGACCTGCTTATAATAGTGACCCAATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCAGGGCAACCTAAGGCTCCATCAGTCTTCCCACTGGCCCCCTGCTGCGGGGACACACCCAGCTCCACGGTGACCCTGGGCTGCCTGGTCAAAGGGTACCTCCCGGAGCCAGTGACCGTGACCTGGAACTCGGGCACCCTCACCAATGGGGTACGCACCTTCCCGTCCGTCCGGCAGTCCTCAGGCCTCTACTCGCTGAGCAGCGTGGTGAGCGTGACCTCAAGCAGCCAGCCCGTCACCTGCAACGTGGCCCACCCAGCCACCAACACCAAAGTGGACAAGACCGTTGCGCCCTCGACATGCAGCAAGCCCACGTGCCCACCCCCTGAACTCCTGGGGGGACCGTCTGTCTTCATCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCACGCACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCAGGATGACCCCGAGGTGCAGTTCACATGGTACATAAACAACGAGCAGGTGCGCACCGCCCGGCCGCCGCTACGGGAGCAGCAGTTCAACAGCACGATCCGCGTGGTCAGCACCCTCCCCATCGCGCACCAGGACTGGCTGAGGGGCAAGGAGTTCAAGTGCAAAGTCCACAACAAGGCACTCCCGGCCCCCATCGAGAAAACCATCTCCAAAGCCAGAGGGCAGCCCCTGGAGCCGAAGGTCTACACCATGGGCCCTCCCCGGGAGGAGCTGAGCAGCAGGTCGGTCAGCCTGACCTGCATGATCAACGGCTTCTACCCTTCCGACATCTCGGTGGAGTGGGAGAAGAACGGGAAGGCAGAGGACAACTACAAGACCACGCCGGCCGTGCTGGACAGCGACGGCTCCTACTTCCTCTACAGCAAGCTCTCAGTGCCCACGAGTGAGTGGCAGCGGGGCGACGTCTTCACCTGCTCCGTGATGCACGAGGCCTTGCACAACCACTACACGCAGAAGTCCATCTCCCGCTCTCCGGGTAAATGA 85-1 Heavy Chain Variable Region Nucleic Acid Sequence: (SEQ ID NO: 63)CAGTCGCTGGAGGAGTCCGGGGGTCACCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCAAAGCCTCTGGATTCTCCCTCAGTAGCTACTGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTGGTGGTATCTGTAGCACATACTACGCAGCCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCGCCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGACCTGCTTATAATAGTGACCCAATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA 85-1 Heavy Chain (H1) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 37)

TVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  85-1 Heavy Chain (H1) Amino Acid Sequence: (SEQ ID NO: 14)

GDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* 85-1 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 64)

Clone 85-1 Light Chain (L3) Nucleic Acid Sequence with 5′ Sequence  Including HindIII Restriction Site (underlined) and Signal Peptide (bold): (SEQ ID NO: 38) AAGCTTGTACCCTTCACCATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGTCCTGATCTATGATGCTGCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAGAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTGGTAGTGCTGATTGTAATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGTGAGAGCGGCCGC Clone 85-1 Light Chain (L3) Nucleic Acid Sequence with Signal Peptide (bold): (SEQ ID NO: 39)ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCACATTTGCCCAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGTCCTGATCTATGATGCTGCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAGAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTGGTAGTGCTGATTGTAATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGTGAGAGCGGCCGCClone 85-1 Light Chain (L3) Nucleic Acid Sequence: (SEQ ID NO: 15)CAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGTCCTGATCTATGATGCTGCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAGAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTGGTAGTGCTGATTGTAATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAGAGTGAGAGCGGCCGC Clone 85-1 Light Chain Variable Region Nucleic Acid Sequence:(SEQ ID NO: 65)CAAGTGCTGACCCAGACTCCATCCCCTGTGTCTGTAGCTGTGGGAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAAGTCCTGATCTATGATGCTGCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAGAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGAATTTAGTTGTGGTAGTGCTGATTGTAATGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA 85-1 Light Chain (L3) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 40)

GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* 85-1 Light Chain (L3) Amino Acid Sequence: (SEQ ID NO: 16)

QVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC* 85-1 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 66)

In some embodiments, antibody 85-1 is modified to remove one or morecysteine residues in one or more CDR sequences. In some embodiments, theantibody includes a CDRH1 sequence that comprises the amino acidsequence SYXMS (SEQ ID NO: 101), where X is Ala or Ser, a CDRH2 sequencethat comprises the amino acid sequence IIGGIXSTYYAAWAKG (SEQ ID NO:102), where X is Ala or Ser, a CDRH3 sequence that comprises the aminoacid sequence PAYNSDPI (SEQ ID NO: 97), a CDRL1 sequence that comprisesthe amino acid sequence QASQSVYNNNYLS (SEQ ID NO: 98), a CDRL2 sequencethat comprises the amino acid sequence DAATLAS (SEQ ID NO: 99), and aCDRL3 sequence that comprises the amino acid sequence LGEFSXGSADXNA (SEQID NO: 103), where X is Ala or Ser. In some embodiments, the antibodyincludes the following heavy and light chain amino acid sequences:

85-1 Heavy Chain (H1) Amino Acid Sequence with Signal Peptide (bold):(SEQ ID NO: 104)

TVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK*  Where X is Ala or Ser85-1 Heavy Chain (H1) Amino Acid Sequence: (SEQ ID NO: 105)

GDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK* Where X is Ala or Ser85-1 Heavy Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 106)

Where X is Ala or Ser85-1 Light Chain (L3) Amino Acid Sequence with Signal Peptide  (bold):(SEQ ID NO: 107)

GTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser85-1 Light Chain (L3) Amino Acid Sequence: (SEQ ID NO: 108)

QVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC*  Where X is Ala or Ser85-1 Light Chain Variable Region Amino Acid Sequence: (SEQ ID NO: 109)

Where X is Ala or Ser

Activatable Antibodies

The antibodies and antigen-binding fragments thereof of the disclosurebind an activatable antibody and/or a conjugated activatable antibodythat binds a target. The activatable antibodies include an antibody orantigen-binding fragment thereof (AB) that specifically binds a targetcoupled to a masking moiety (MM), such that coupling of the MM reducesthe ability of the antibody or antigen-binding fragment thereof to bindthe target. In some embodiments, the MM is coupled to the AB via asequence that includes a substrate for a protease, for example, aprotease that is co-localized with the target at a treatment site in asubject.

The conjugated activatable antibodies include an antibody orantigen-binding fragment thereof (AB) that specifically binds a targetcoupled to a masking moiety (MM), such that coupling of the MM reducesthe ability of the antibody or antigen-binding fragment thereof to bindthe target, and an agent conjugated or otherwise coupled to theactivatable antibody. In some embodiments, the MM is coupled to the ABvia a sequence that includes a substrate for a protease, for example, aprotease that is co-localized with the target at a treatment site in asubject.

The conjugated antibodies and/or activatable antibodies include anantibody or antigen-binding fragment thereof (AB) that specificallybinds a target. Exemplary classes of targets of an AB include, but arenot necessarily limited to, cell surface receptors and secreted bindingproteins (e.g., growth factors), soluble enzymes, structural proteins(e.g. collagen, fibronectin) and the like. In some embodiments,conjugated antibodies and/or activatable antibodies have an AB thatbinds an extracellular target, usually an extracellular protein target.In other embodiments, conjugated antibodies and/or activatableantibodies are designed for cellular uptake such that the conjugatedantibodies and/or activatable antibodies are activated inside a cell.

As a non-limiting example, the AB is a binding partner for any targetlisted in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 CD52 DL44 HVEM LIF-R STEAP1 Alpha-4CD56 DLK1 Hyaluronidase Lewis X STEAP2 integrin Alpha-V CD64 DLL4 ICOSLIGHT TAG-72 integrin alpha4beta1 CD70 DPP-4 IFNalpha LRP4 TAPA1integrin alpha4beta7 CD71 DSG1 IFNbeta LRRC26 TGFbeta integrin AGR2 CD74EGFR IFNgamma MCSP TIGIT Anti-Lewis-Y EGFRviii IgE Mesothelin TIM-3Apelin J CD80 Endothelin B IgE Receptor MRP4 TLR2 receptor receptor(FceRI) (ETBR) APRIL CD81 ENPP3 IGF MUC1 TLR4 B7-H4 CD86 EpCAM IGF1RMucin-16 TLR6 (MUC16, CA-125) BAFF CD95 EPHA2 IL1B Na/K ATPase TLR7 BTLACD117 EPHB2 IL1R Neutrophil TLR8 elastase C5 CD125 ERBB3 IL2 NGF TLR9complement C-242 CD132 F protein of IL11 Nicastrin TMEM31 (IL-2RG) RSVCA9 CD133 FAP IL12 Notch TNFalpha Receptors CA19-9 CD137 FGF-2 IL12p40Notch 1 TNFR (Lewis a) Carbonic CD138 FGF8 IL-12R, Notch 2 TNFRS12Aanhydrase 9 IL-12Rbeta1 CD2 CD166 FGFR1 IL13 Notch 3 TRAIL-R1 CD3 CD172AFGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3 IL15 NOV Transferrin CD9CDH6 FGFR4 IL17 OSM-R Transferrin receptor CD11a CEACAM5 Folate IL18OX-40 TRK-A (CEA) receptor CD19 CEACAM6 GAL3ST1 IL21 PAR2 TRK-B (NCA-90)CD20 CLAUDIN-3 G-CSF IL23 PDGF-AA uPAR CD22 CLAUDIN-4 G-CSFR IL23RPDGF-BB VAP1 CD24 cMet GD2 IL27/IL27R PDGFRalpha VCAM-1 (wsx1) CD25Collagen GITR IL29 PDGFRbeta VEGF CD27 Cripto GLUT1 IL-31R PD-1 VEGF-ACD28 CSFR GLUT4 IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2VEGF-C CD33 CTLA-4 GM-CSFR IL4 Phosphatidyl- VEGF-D serine CD38 CTGF GPIIb/IIIa IL4R P1GF VEGFR1 receptors CD40 CXCL10 Gp130 IL6, IL6R PSCAVEGFR2 CD40L CXCL13 GPIIB/IIIA Insulin PSMA VEGFR3 Receptor CD41 CXCR1GPNMB Jagged RAAG12 VISTA Ligands CD44 CXCR2 GRP78 Jagged 1 RAGE WISP-1CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47 CXCR4 HGF LAG-3 Sphingosine1 WISP-3 Phosphate CD51 CYR61 hGH

As a non-limiting example, the AB is or is derived from an antibodylisted in Table 2.

TABLE 2 Exemplary sources for ABs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ CD20 (ibritumomab tiuxetan) Rituxan ™ (rituximab) CD20Ocrelizumab CD20 Arzerra ™ (ofatumumab) CD20 Obinutuzumab CD20 Zenapax ™(daclizumab) CD25 Adcetris ™ CD30 (brentuximab vedotin) Myelotarg ™(gemtuzumab) CD33 Mylotarg ™ CD33 (gemtuzumab ozogamicin) Campath ™(alemtuzumab) CD52 ReoPro ™ (abiciximab) Glycoprotein receptor IIb/IIIaXolair ™ (omalizumab) IgE Herceptin ™ (trastuzumab) Her2 Kadcyla ™ Her2(trastuzumab emtansine) Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab)Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4(tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR(mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3Antril ™, Kineret ™ IL-1Ra (ankinra) GC1008 TGFbeta Notch, e.g., Notch 1Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R(tocilizumab) IL-6 receptor Stelara ™ (ustekinumab) IL-12/IL-23 Prolia ™(denosumab) RANKL

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds the target. Insome embodiments, the antibody or immunologically active fragmentthereof is a monoclonal antibody, domain antibody, single chain, Fabfragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody. In someembodiments, such an antibody or immunologically active fragment thereofis a rabbit, mouse, chimeric, humanized or fully human monoclonalantibody.

The activatable antibodies and activatable antibody compositionsprovided herein contain at least an antibody or antibody fragmentthereof (collectively referred to as AB throughout the disclosure) thatspecifically binds a target, e.g., a human target, wherein the AB ismodified by a masking moiety (MM).

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is no more than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM does not interfere or compete with the AB ofthe activatable antibody in a cleaved state for binding to the target.

In some embodiments, the masking moiety is selected for use with aspecific antibody or antibody fragment. For example, suitable maskingmoieties for use with antibodies that bind EGFR include MMs that includethe sequence CISPRG (SEQ ID NO: 111). By way of non-limiting examples,the MM can include a sequence such as CISPRGCG (SEQ ID NO: 112);CISPRGCPDGPYVMY (SEQ ID NO: 113); CISPRGCPDGPYVM (SEQ ID NO: 114),CISPRGCEPGTYVPT (SEQ ID NO: 115) and CISPRGCPGQIWHPP (SEQ ID NO: 116).Other suitable masking moieties include any of the EGFR-specific masksdisclosed in PCT Publication No. WO 2010/081173, such as, by way ofnon-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 117);CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 118); CNHHYFYTCGCISPRGCPG(SEQ ID NO: 119); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 120);CHHVYWGHCGCISPRGCPG (SEQ ID NO: 121); CPHFTTTSCGCISPRGCPG (SEQ ID NO:122); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 123); CPHVSFGSCGCISPRGCPG (SEQ IDNO: 124); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 125); CNHVYFGTCGCISPRGCPG (SEQID NO: 126); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 127); CHHFTLTTCGCISPRGCPG(SEQ ID NO: 128); YNPCATPMCCISPRGCPG (SEQ ID NO: 129);CNHHYFYTCGCISPRGCG (SEQ ID NO: 130); CNHHYHYYCGCISPRGCG (SEQ ID NO:131); CNHVYFGTCGCISPRGCG (SEQ ID NO: 132); CHHVYWGHCGCISPRGCG (SEQ IDNO: 133); CPHFTTTSCGCISPRGCG (SEQ ID NO: 134); CNHFTLTTCGCISPRGCG (SEQID NO: 135); CHHFTLTTCGCISPRGCG (SEQ ID NO: 136); CPYYTLSYCGCISPRGCG(SEQ ID NO: 137); CPHVSFGSCGCISPRGCG (SEQ ID NO: 138);ADHVFWGSYGCISPRGCG (SEQ ID NO: 139); YNPCATPMCCISPRGCG (SEQ ID NO: 140);CHHVYWGHCGCISPRGCG (SEQ ID NO: 141);C(N/P)H(HN/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO:142); CISPRGCGQPIPSVK (SEQ ID NO: 143); CISPRGCTQPYHVSR (SEQ ID NO:144); and/or CISPRGCNAVSGLGS (SEQ ID NO: 145).

Suitable masking moieties for use with antibodies that bind a Jaggedtarget, e.g., Jagged 1 and/or Jagged 2, include, by way of non-limitingexample, masking moieties that include a sequence such asQGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 146); PWCMQRQDFLRCPQP (SEQ ID NO:147); QLGLPAYMCTFECLR (SEQ ID NO: 148); CNLWVSGGDCGGLQG (SEQ ID NO:149); SCSLWTSGSCLPHSP (SEQ ID NO: 150); YCLQLPHYMQAMCGR (SEQ ID NO:151); CFLYSCTDVSYWNNT (SEQ ID NO: 152); PWCMQRQDYLRCPQP (SEQ ID NO:153); CNLWISGGDCRGLAG (SEQ ID NO: 154); CNLWVSGGDCRGVQG (SEQ ID NO:155); CNLWVSGGDCRGLRG (SEQ ID NO: 156); CNLWISGGDCRGLPG (SEQ ID NO:157); CNLWVSGGDCRDAPW (SEQ ID NO: 158); CNLWVSGGDCRDLLG (SEQ ID NO:159); CNLWVSGGDCRGLQG (SEQ ID NO: 160); CNLWLHGGDCRGWQG (SEQ ID NO:161); CNIWLVGGDCRGWQG (SEQ ID NO: 162); CTTWFCGGDCGVMRG (SEQ ID NO:163); CNIWGPSVDCGALLG (SEQ ID NO: 164); CNIWVNGGDCRSFEG (SEQ ID NO:165); YCLNLPRYMQDMCWA (SEQ ID NO: 166); YCLALPHYMQADCAR (SEQ ID NO:167); CFLYSCGDVSYWGSA (SEQ ID NO: 168); CYLYSCTDSAFWNNR (SEQ ID NO:169); CYLYSCNDVSYWSNT (SEQ ID NO: 170); CFLYSCTDVSYW (SEQ ID NO: 171);CFLYSCTDVAYWNSA (SEQ ID NO: 172); CFLYSCTDVSYWGDT (SEQ ID NO: 173);CFLYSCTDVSYWGNS (SEQ ID NO: 174); CFLYSCTDVAYWNNT (SEQ ID NO: 175);CFLYSCGDVSYWGNPGLS (SEQ ID NO: 176); CFLYSCTDVAYWSGL (SEQ ID NO: 177);CYLYSCTDGSYWNST (SEQ ID NO: 178); CFLYSCSDVSYWGNI (SEQ ID NO: 179);CFLYSCTDVAYW (SEQ ID NO: 180); CFLYSCTDVSYWGST (SEQ ID NO: 181);CFLYSCTDVAYWGDT (SEQ ID NO: 182); GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 183);GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 184); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO:185); GCNIWANGGDCRGWIDNIDG (SEQ ID NO: 186); GCNIWLVGGDCRGWLGEAVG (SEQID NO: 187); GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 188); GGPALCNIWLNGGDCRGWSG(SEQ ID NO: 189); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 190);GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 191); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO:192); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 193); GASQYCNLWINGGDCRGWRG (SEQID NO: 194); GCNIWLVGGDCRPWVEGG (SEQ ID NO: 195); GCNIWAVGGDCRPFVDGG(SEQ ID NO: 196); GCNIWLNGGDCRAWVDTG (SEQ ID NO: 197);GCNIWIVGGDCRPFINDG (SEQ ID NO: 198); GCNIWLNGGDCRPVVFGG (SEQ ID NO:199); GCNIWLSGGDCRMFMNEG (SEQ ID NO: 200); GCNIWVNGGDCRSFVYSG (SEQ IDNO: 201); GCNIWLNGGDCRGWEASG (SEQ ID NO: 202); GCNIWAHGGDCRGFIEPG (SEQID NO: 203); GCNIWLNGGDCRTFVASG (SEQ ID NO: 204); GCNIWAHGGDCRGFIEPG(SEQ ID NO: 205); GFLENCNIWLNGGDCRTG (SEQ ID NO: 206);GIYENCNIWLNGGDCRMG (SEQ ID NO: 207); and/or GIPDNCNIWINGGDCRYG (SEQ IDNO: 208).

Suitable masking moieties for use with antibodies that bind aninterleukin 6 target, e.g., interleukin 6 receptor (IL-6R), include, byway of non-limiting example, masking moieties that include a sequencesuch as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 209); QGQSGQGDFDIPFPAHWVPIT(SEQ ID NO: 210); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 211);YRSCNWNYVSIFLDC (SEQ ID NO: 212); PGAFDIPFPAHWVPNT (SEQ ID NO: 213);ESSCVWNYVHIYMDC (SEQ ID NO: 214); YPGCKWNYDRIFLDC (SEQ ID NO: 215);YRTCSWNYVGIFLDC (SEQ ID NO: 216); YGSCSWNYVHIFMDC (SEQ ID NO: 217);YGSCSWNYVHIFLDC (SEQ ID NO: 218); YGSCNWNYVHIFLDC (SEQ ID NO: 219);YTSCNWNYVHIFMDC (SEQ ID NO: 220); YPGCKWNYDRIFLDC (SEQ ID NO: 221);WRSCNWNYAHIFLDC (SEQ ID NO: 222); WSNCHWNYVHIFLDC (SEQ ID NO: 223);DRSCTWNYVRISYDC (SEQ ID NO: 224); SGSCKWDYVHIFLDC (SEQ ID NO: 225);SRSCIWNYAHIHLDC (SEQ ID NO: 226); SMSCYWQYERIFLDC (SEQ ID NO: 227);YRSCNWNYVSIFLDC (SEQ ID NO: 228); SGSCKWDYVHIFLDC (SEQ ID NO: 229);YKSCHWDYVHIFLDC (SEQ ID NO: 230); YGSCTWNYVHIFMEC (SEQ ID NO: 231);FSSCNWNYVHIFLDC (SEQ ID NO: 232); WRSCNWNYAHIFLDC (SEQ ID NO: 233);YGSCQWNYVHIFLDC (SEQ ID NO: 234); YRSCNWNYVHIFLDC (SEQ ID NO: 235);NMSCHWDYVHIFLDC (SEQ ID NO: 236); FGPCTWNYARISWDC (SEQ ID NO: 237);XXsCXWXYvhIfXdC (SEQ ID NO: 238); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 239);RDTGGQCRWDYVHIFMDC (SEQ ID NO: 240); AGVPAGCTWNYVHIFMEC (SEQ ID NO:241); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 242); DGGPAGCSWNYVHIFMEC (SEQ IDNO: 243); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 244); CTWNYVHIFMDCGEGEGP (SEQID NO: 245); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 246); AEVPAGCWWNYVHIFMEC(SEQ ID NO: 247); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 248);SGASGGCKWNYVHIFMDC (SEQ ID NO: 249); TPGCRWNYVHIFMECEAL (SEQ ID NO:250); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 251); PGAFDIPFPAHWVPNT (SEQ ID NO:252); RGACDIPFPAHWIPNT (SEQ ID NO: 253); QGDFDIPFPAHWVPIT (SEQ ID NO:254); XGafDIPFPAHWvPnT (SEQ ID NO: 255); RGDGNDSDIPFPAHWVPRT (SEQ ID NO:256); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 257); WAGGNDCDIPFPAHWIPNT (SEQ IDNO: 258); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 259); AGSGNDSDIPFPAHWVPRT (SEQID NO: 260); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 261); and/orRECGRCGDIPFPAHWVPRT (SEQ ID NO: 262).

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody includes a linking peptidebetween the MM and the CM, i.e., the substrate sequence.

In some embodiments, the activatable antibody includes a linking peptidebetween the CM and the AB.

In some embodiments, the activatable antibody includes a first linkingpeptide (LP1) and a second linking peptide (LP2), and the activatableantibody in the uncleaved state has the structural arrangement fromN-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB orAB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need notbe identical to each other.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length, for example, no more than 40 amino acids long.

In some embodiments, the MM polypeptide sequence is different from thatof the target, and the MM polypeptide sequence is no more than 50%identical to any natural binding partner of the AB. In some embodiments,the MM does not include more than 25% amino acid sequence identity tothe target. In some embodiments, the MM does not include more than 10%amino acid sequence identity to the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least twofold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least five-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least ten-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 20-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 40-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 100-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is reduced to occur with an equilibrium dissociation constantthat is at least 200-fold greater than the equilibrium dissociationconstant of an unmodified AB binding to the target, and whereas the ABof the activatable antibody in the cleaved state binds the target.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind the target such that the dissociation constant (K_(d)) of theAB when coupled to the MM towards the target is at least two timesgreater than the K_(d) of the AB when not coupled to the MM towards thetarget. In some embodiments, the coupling of the MM reduces the abilityof the AB to bind the target such that the dissociation constant (K_(d))of the AB when coupled to the MM towards the target is at least fourtimes greater than the K_(d) of the AB when not coupled to the MMtowards the target. In some embodiments, the coupling of the MM reducesthe ability of the AB to bind the target such that the dissociationconstant (K_(d)) of the AB when coupled to the MM towards the target isat least five times greater than the K_(d) of the AB when not coupled tothe MM towards the target. In some embodiments, the coupling of the MMreduces the ability of the AB to bind the target such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsthe target is at least 10 times greater than the K_(d) of the AB whennot coupled to the MM towards the target. In some embodiments, thecoupling of the MM reduces the ability of the AB to bind the target suchthat the dissociation constant (K_(d)) of the AB when coupled to the MMtowards the target is at least 20 times greater than the K_(d) of the ABwhen not coupled to the MM towards the target. In some embodiments, thecoupling of the MM reduces the ability of the AB to bind the target suchthat the K_(d) of the AB when coupled to the MM towards the target is atleast 40 times greater than the K_(d) of the AB when not coupled to theMM towards the target. In some embodiments, the coupling of the MMreduces the ability of the AB to bind the target such that the K_(d) ofthe AB when coupled to the MM towards the target is at least 100 timesgreater than the K_(d) of the AB when not coupled to the MM towards thetarget. In some embodiments, the coupling of the MM reduces the abilityof the AB to bind the target such that the K_(d) of the AB when coupledto the MM towards the target is at least 1000 times greater than theK_(d) of the AB when not coupled to the MM towards the target. In someembodiments, the coupling of the MM reduces the ability of the AB tobind the target such that the K_(d) of the AB when coupled to the MMtowards the target is at least 10,000 times greater than the K_(d) ofthe AB when not coupled to the MM towards the target.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength. The CM is cleaved by a protease. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the protease is notactive or is significantly less active in tissues that do notsignificantly express the target. In some embodiments, the protease isnot active or is significantly less active in healthy, e.g.,non-diseased tissues.

In some embodiments, the CM is cleaved by a protease shown in Table 3.

TABLE 3 Exemplary proteases ADAMS, ADAMTS, e.g. ADAM8  ADAM9  ADAM10ADAM12 ADAM15 ADAM17/TACE ADAMDEC1 ADAMTS1 ADAMTS4 ADAMTS5 Aspartateproteases, e.g., BACE Renin Aspartic cathepsins, e.g., Cathepsin DCathepsin E Caspases, e.g., Caspase 1  Caspase 2  Caspase 3  Caspase 4 Caspase 5  Caspase 6  Caspase 7  Caspase 8  Caspase 9  Caspase 10Caspase 14 Cysteine cathepsins, e.g., Cathepsin B Cathepsin C CathepsinK Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/P Cysteineproteinases, e.g., Cruzipain Legumain Otubain-2 KLKs, e.g., KLK4  KLK5 KLK6  KLK7  KLK8  KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g.,Meprin Neprilysin PSMA BMP-1 MMPs, e.g., MMP1  MMP2  MMP3  MMP7  MMP8 MMP9  MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP23MMP24 MMP26 MMP27 Serine proteases, e.g., activated protein C CathepsinA Cathepsin G Chymase coagulation factor proteases (e.g., FVIIa, FIXa,FXa, FXIa, FXIIa) Elastase Granzyme B Guanidinobenzoatase HtrA1 HumanNeutrophil Elastase Lactoferrin Marapsin NS3/4A PACE4 Plasmin PSA tPAThrombin Tryptase uPA Type II Transmembrane Serine Proteases (TTSPs),e.g., DESC1 DPP-4 FAP Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2TMPRSS3 TMPRSS4

In some embodiments, the CM is selected for use with a specificprotease. In some embodiments, the CM is a substrate for at least oneprotease selected from the group consisting of an ADAM 17, a BMP-1, acysteine protease such as a cathepsin, a HtrAl, a legumain, a matriptase(MT-SP1), a matrix metalloprotease (MMP), a neutrophil elastase, aTMPRSS, such as TMPRSS3 or TMPRSS4, a thrombin, and a u-type plasminogenactivator (uPA, also referred to as urokinase),

In some embodiments, the CM is a substrate for an ADAM17. In someembodiments, the CM is a substrate for a BMP-1. In some embodiments, theCM is a substrate for a cathepsin. In some embodiments, the CM is asubstrate for a cysteine protease. In some embodiments, the CM is asubstrate for a HtrA1. In some embodiments, the CM is a substrate for alegumain. In some embodiments, the CM is a substrate for a matriptase.In some embodiments, the CM is a substrate for a MMP. In someembodiments, the CM is a substrate for a neutrophil elastase. In someembodiments, the CM is a substrate for a thrombin. In some embodiments,the CM is a substrate for a TMPRSS. In some embodiments, the CM is asubstrate for TMPRSS3. In some embodiments, the CM is a substrate forTMPRSS4. In some embodiments, the CM is a substrate for uPA.

For example, suitable CM are cleaved by at least one protease andinclude the sequence TGRGPSWV (SEQ ID NO: 263); SARGPSRW (SEQ ID NO:264); TARGPSFK (SEQ ID NO: 265); LSGRSDNH (SEQ ID NO: 266); GGWHTGRN(SEQ ID NO: 267); HTGRSGAL (SEQ ID NO: 268); PLTGRSGG (SEQ ID NO: 269);AARGPAIH (SEQ ID NO: 270); RGPAFNPM (SEQ ID NO: 271); SSRGPAYL (SEQ IDNO: 272); RGPATPIM (SEQ ID NO: 273); RGPA (SEQ ID NO: 274); GGQPSGMWGW(SEQ ID NO: 275); FPRPLGITGL (SEQ ID NO: 276); VHMPLGFLGP (SEQ ID NO:277); SPLTGRSG (SEQ ID NO: 278); SAGFSLPA (SEQ ID NO: 279); LAPLGLQRR(SEQ ID NO: 280); SGGPLGVR (SEQ ID NO: 281); PLGL (SEQ ID NO: 282);GPRSFGL (SEQ ID NO: 283) and/or GPRSFG (SEQ ID NO: 284).

In some embodiments, the CM comprises the amino acid sequence TGRGPSWV(SEQ ID NO: 263). In some embodiments, the CM comprises the amino acidsequence SARGPSRW (SEQ ID NO: 264). In some embodiments, the CMcomprises the amino acid sequence TARGPSFK (SEQ ID NO: 265). In someembodiments, the CM comprises the amino acid sequence LSGRSDNH (SEQ IDNO: 266). In some embodiments, the CM comprises the amino acid sequenceGGWHTGRN (SEQ ID NO: 267). In some embodiments, the CM comprises theamino acid sequence HTGRSGAL (SEQ ID NO: 268). In some embodiments, theCM comprises the amino acid sequence PLTGRSGG (SEQ ID NO: 269). In someembodiments, the CM comprises the amino acid sequence AARGPAIH (SEQ IDNO: 270). In some embodiments, the CM comprises the amino acid sequenceRGPAFNPM (SEQ ID NO: 271). In some embodiments, the CM comprises theamino acid sequence SSRGPAYL (SEQ ID NO: 272). In some embodiments, theCM comprises the amino acid sequence RGPATPIM (SEQ ID NO: 273). In someembodiments, the CM comprises the amino acid sequence RGPA (SEQ ID NO:274). In some embodiments, the CM comprises the amino acid sequenceGGQPSGMWGW (SEQ ID NO: 275). In some embodiments, the CM comprises theamino acid sequence FPRPLGITGL (SEQ ID NO: 276). In some embodiments,the CM comprises the amino acid sequence VHMPLGFLGP (SEQ ID NO: 277). Insome embodiments, the CM comprises the amino acid sequence SPLTGRSG (SEQID NO: 278). In some embodiments, the CM comprises the amino acidsequence SAGFSLPA (SEQ ID NO: 279). In some embodiments, the CMcomprises the amino acid sequence LAPLGLQRR (SEQ ID NO: 280). In someembodiments, the CM comprises the amino acid sequence SGGPLGVR (SEQ IDNO: 281). In some embodiments, the CM comprises the amino acid sequencePLGL (SEQ ID NO: 282). In some embodiments, the CM comprises the aminoacid sequence GPRSFGL (SEQ ID NO: 283). In some embodiments, the CMcomprises the amino acid sequence GPRSFG (SEQ ID NO: 284).

In some embodiments, the CM is a substrate for at least one MMP. In someembodiments, the CM is a substrate for at least one MMP listed in theTable 3. In some embodiments the CM1 is a substrate for MMP9. In someembodiments, the CM1 is a substrate for MMP14. In some embodiments, theCM is a substrate for two or more MMPs. In some embodiments, the CM is asubstrate for at least MMP9 or MMP14. In some embodiments, the CM is asubstrate for two or more MMPs.

In some embodiments, the CM is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 45); QNQALRMA (SEQ ID NO: 46); AQNLLGMV(SEQ ID NO: 47); STFPFGMF (SEQ ID NO: 48); PVGYTSSL (SEQ ID NO: 49);DWLYWPGI (SEQ ID NO: 50) MIAPVAYR (SEQ ID NO: 51); RPSPMWAY (SEQ ID NO:52); WATPRPMR (SEQ ID NO: 53); FRLLDWQW (SEQ ID NO: 54); LKAAPRWA (SEQID NO: 398); GPSHLVLT (SEQ ID NO: 399); LPGGLSPW (SEQ ID NO: 400);MGLFSEAG (SEQ ID NO: 401); SPLPLRVP (SEQ ID NO: 402); RMHLRSLG (SEQ IDNO: 403); LAAPLGLL (SEQ ID NO: 404); AVGLLAPP (SEQ ID NO: 405); LLAPSHRA(SEQ ID NO: 406), PAGLWLDP (SEQ ID NO: 407); and/or ISSGLSS (SEQ ID NO:408).

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 285) and (GGGS)_(n) (SEQ ID NO: 286), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 287), GGSGG (SEQ ID NO: 288), GSGSG (SEQ ID NO:289), GSGGG (SEQ ID NO: 290), GGGSG (SEQ ID NO: 291), and GSSSG (SEQ IDNO: 292).

In some embodiments, LP1 includes the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 293).

In some embodiments, LP2 includes the amino acid sequence GSSGT (SEQ IDNO: 294) or GSSG (SEQ ID NO: 295).

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. As usedherein, a fragment of a toxin is a fragment that retains toxic activity.In some embodiments, the agent is conjugated to the AB via a cleavablelinker. In some embodiments, the agent is conjugated to the AB via alinker that includes at least one MMP-cleavable substrate sequence. Insome embodiments, the agent is conjugated to the AB via a linker thatincludes at least one cathepsin-cleavable substrate sequence. In someembodiments, the agent is conjugated to the AB via a noncleavablelinker. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a nucleic acid damaging agent, such as aDNA alkylator or DNA intercalator, or other DNA damaging agent. In someembodiments, the agent is an agent selected from the group listed inTable 4. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is monomethyl auristatin D (MMAD). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof. In some embodiments, theagent is a pyrrolobenzodiazepine (PBD).

In some embodiments, the agent is conjugated to the AB via a linker. Insome embodiments, the linker is a cleavable linker.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent. In some embodiments, the detectable moiety is a conjugatabledetection reagent. In some embodiments, the detectable moiety is, forexample, a fluorescein derivative such as fluorescein isothiocyanate(FITC).

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer includes atleast the amino acid sequence QGQSGQ (SEQ ID NO: 296). In someembodiments, an activatable antibody includes a spacer of sequenceQGQSGQ (SEQ ID NO: 296) joined directly to a MM sequence in thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.

In some embodiments, the serum half-life of the activatable antibodyand/or conjugated activatable antibody is longer than that of thecorresponding antibody; e.g., the pK of the activatable antibody and/orconjugated activatable antibody is longer than that of the correspondingantibody. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is similar to that ofthe corresponding antibody. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 15 days when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 11 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 10 days when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 9 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 8 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 7 days when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 6 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 5 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 4 days when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 3 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 2 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 24 hours when administered to an organism. In some embodiments,the serum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 20 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 18 hourswhen administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody and/or conjugated activatableantibody is at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody and/orconjugated activatable antibody is at least 14 hours when administeredto an organism. In some embodiments, the serum half-life of theactivatable antibody and/or conjugated activatable antibody is at least12 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 8 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody and/or conjugated activatable antibody is atleast 6 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody and/or conjugatedactivatable antibody is at least 4 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody and/or conjugated activatable antibody is at least 3 hours whenadministered to an organism.

In some embodiments, the activatable antibody is monospecific. In someembodiments, the activatable antibody is multispecific, e.g., by way ofnon-limiting example, bispecific or trifunctional. In some embodiments,the activatable antibody is formulated as part of a pro-Bispecific TCell Engager (BITE) molecule. In some embodiments, the activatableantibody is formulated as part of a pro-Chimeric Antigen Receptor (CAR)modified T cell or other engineered receptor.

Exemplary activatable anti-EGFR antibodies of the invention include, forexample, the activatable antibody referred to herein as the3954-1204-C225v5 activatable antibody, which binds epidermal growthfactor receptor (EGFR) when the activatable antibody is in an activated,i.e., cleaved, state. Three sequences of the 3954-1204-C225v5activatable anti-EGFR antibody are shown below, Sequence 1 is thesequence of a version of the 3954-1204-C225v5 activatable anti-EGFRantibody that includes a signal peptide, Sequence 2 is the sequence ofthe 3954-1204-C225v5 activatable anti-EGFR antibody without the signalpeptide, and Sequence 3 is the sequence of the 3954-1204-C225v5activatable anti-EGFR antibody without the signal peptide and withoutthe spacer sequence:

3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][C225v5 (SEQ ID NO: 298)](SEQ ID NO: 299)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence 3954-1204-C225v5 Activatable Antibody Heavy ChainAmino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][C225v5 (SEQ ID NO: 301)](SEQ ID NO: 302)[MYRMQLLSCIALSLALVTNS][QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNIDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*] Italics: Signal peptide Normal text:anti-EGFR antibody derived  sequence3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204 Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 309)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac] [ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1 Bold and Underline:1204 Substrate Bold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204 Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 311)[MYRMQLLSCIALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Italics: Signal peptide Bold:Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v5 (SEQ ID NO: 298)] (SEQ ID NO: 298)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v5 Activatable Antibody Heavy ChainAmino Acid Sequence 2: [C225v5 (SEQ ID NO: 301)] (SEQ ID NO: 301)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204 Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 312)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtacggctcgagcggtggcagcggtggctctggtggatccggt ] [ ctgagcggccgttccgataat cat][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204 Substrate (SEQ ID NO: 266)][Linker  2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 313)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and Underline:Linker 1 Bold and Underline: 1204 Substrate Bold, Italics and Underline:Linker 2 Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence 3:[C225v5 (SEQ ID NO: 298)] (SEQ ID NO: 298)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v5 Activatable Antibody Heavy ChainAmino Acid Sequence 3: [C225v5 (SEQ ID NO: 301)] (SEQ ID NO: 301)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence 3:[Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 409)[tgcatctcacctcgtggttgtccggacggcccatacgtcatgtacggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataatcat ][

] [cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence 3:[Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 410) [CISPRGCPDGPYVMY][GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Another exemplary activatable anti-EGFR antibody of the invention is theactivatable antibody referred to herein as the 3954-1204-C225v4activatable antibody, which binds epidermal growth factor receptor(EGFR) when the activatable antibody is in an activated state. Threesequences of the 3954-1204-C225v4 activatable anti-EGFR antibody areshown below, Sequence 1 is the sequence of a version of the3954-1204-C225v4 activatable anti-EGFR antibody that includes a signalpeptide, Sequence 2 is the sequence of the 3954-1204-C225v4 activatableanti-EGFR antibody without the signal peptide, and Sequence 3 is thesequence of the 3954-1204-C225v4 activatable anti-EGFR antibody withoutthe signal peptide and without the spacer sequence:

3954-1204-C225v4 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal Peptide (SEQ ID NO: 297)][C225v4 (SEQ ID NO: 314)](SEQ ID NO: 315)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal Peptide (SEQ ID NO: 300)][C225v4 (SEQ ID NO: 316)](SEQ ID NO: 317)[MYRMQLLSCIALSLALVTNS][QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*] Italics: Signal peptide Normal text:anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 318)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac] [ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat ]

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1 Bold and Underline:1204 Substrate Bold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204  Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 319)[MYRMQLLSCIALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Italics: Signal peptide Bold:Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v4 (SEQ ID NO: 314)] (SEQ ID NO: 314)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v4 Activatable Antibody Heavy ChainAmino Acid Sequence 2: [C225v4 (SEQ ID NO: 316)] (SEQ ID NO: 316)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v4 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204 Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 320)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204 Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 321)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLIQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and Underline:Linker 1 Bold and Underline: 1204 Substrate Bold, Italics and Underline:Linker 2 Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Heavy Chain Nucleotide Sequence 3:[C225v4 (SEQ ID NO: 314)] (SEQ ID NO: 314)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v4 Activatable Antibody Heavy ChainAmino Acid Sequence 3: [C225v4 (SEQ ID NO: 316)] (SEQ ID NO: 316)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v4 Activatable Antibody Light Chain Nucleotide Sequence 3:[Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 411)[tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac][ ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataatcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Amino Acid Sequence3:[Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 412) [CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Another exemplary activatable anti-EGFR antibody of the invention is theactivatable antibody referred to herein as the 3954-1204-C225v6activatable antibody, which binds epidermal growth factor receptor(EGFR) when the activatable antibody is in an activated state. Threesequences of the 3954-1204-C225v6 activatable anti-EGFR antibody areshown below, Sequence 1 is the sequence of a version of the3954-1204-C225v6 activatable anti-EGFR antibody that includes a signalpeptide, Sequence 2 is the sequence of the 3954-1204-C225v6 activatableanti-EGFR antibody without the signal peptide, and Sequence 3 is thesequence of the 3954-1204-C225v6 activatable anti-EGFR antibody withoutthe signal peptide and without the spacer sequence:

3954-1204-C225v6 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][C225v6 (SEQ ID NO: 322)](SEQ ID NO: 323)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][C225v6 (SEQ ID NO: 324)](SEQ ID NO: 325)[MYRMQLLSCIALSLALVTNS][QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*] Italics: Signal peptide Normal text:anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 326)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac] [ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1 Bold and Underline:1204 Substrate Bold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 327)[MYRMQLLSCIALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGS GGSG][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Italics: Signal peptide Bold:Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v6 (SEQ ID NO: 322)] (SEQ ID NO: 322)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v6 Activatable Antibody Heavy ChainAmino Acid Sequence 2: [C225v6 (SEQ ID NO: 324)] (SEQ ID NO: 324)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v6 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1(SEQ ID NO: 305)][1204 Substrate (SEQ ID NO: 306)][Linker 2(SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 328)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1(SEQ ID NO: 293)][1204 Substrate (SEQ ID NO: 266)][Linker 2(SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 329)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and Underline:Linker 1 Bold and Underline: 1204 Substrate Bold, Italics and Underline:Linker 2 Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Heavy Chain Nucleotide Sequence 3:[C225v6 (SEQ ID NO: 322)] (SEQ ID NO: 322)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v6 Activatable Antibody Heavy ChainAmino Acid Sequence 3: [C225v6 (SEQ ID NO: 324)] (SEQ ID NO: 324)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v6 Activatable Antibody Light Chain Nucleotide Sequence 3:[Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 413)[tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac][ ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataatcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Amino Acid Sequence 3:[Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 414) [CISPRGCPDGPYVMY][GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Another exemplary activatable anti-EGFR antibody of the invention is theactivatable antibody referred to herein as the 3954-1204-C225v7activatable antibody, which binds epidermal growth factor receptor(EGFR) when the activatable antibody is in an activated state. Threesequences of the 3954-1204-C225v7 activatable anti-EGFR antibody areshown below, Sequence 1 is the sequence of a version of the3954-1204-C225v7 activatable anti-EGFR antibody that includes a signalpeptide, Sequence 2 is the sequence of the 3954-1204-C225v7 activatableanti-EGFR antibody without the signal peptide, and Sequence 3 is thesequence of the 3954-1204-C225v7 activatable anti-EGFR antibody withoutthe signal peptide and without the spacer sequence:

3954-1204-C225v7 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][C225v7 (SEQ ID NO: 425)](SEQ ID NO: 426)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaccaragcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][C225v7 (SEQ ID NO: 427)](SEQ ID NO: 428)[MYRMQLLSCIALSLALVTNS][QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*] Italics: Signal peptide Normal text:anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 297)][Spacer (SEQ ID NO: 303)][Mask(SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204 Substrate(SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)](SEQ ID NO: 326)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac] [ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1 Bold and Underline:1204 Substrate Bold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 300)][Spacer (SEQ ID NO: 296)][Mask(SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204 Substrate (SEQID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)](SEQ ID NO: 327) [MYRMQLLSCIALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGS GGSG][ L SGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Italics: Signal peptide Bold:Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Heavy Chain Nucleotide Sequence 2: [C225v7 (SEQ ID NO: 425)] (SEQ ID NO: 425)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaccaragcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v7 Activatable Antibody Heavy Chain Amino Acid Sequence 2: [C225v7 (SEQ ID NO: 427)] (SEQ ID NO: 427)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v7 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 303)][Mask (SEQ ID NO: 304)][Linker 1 (SEQID NO: 305)][1204 Substrate (SEQ ID NO: 306)][Linker 2 (SEQ IDNO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 328)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2 Normal text:anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 296)][Mask (SEQ ID NO: 113)][Linker 1 (SEQID NO: 293)][1204 Substrate (SEQ ID NO: 266)][Linker 2 (SEQ IDNO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 329)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and Underline:Linker 1 Bold and Underline: 1204 Substrate Bold, Italics and Underline:Linker 2 Normal text: anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Heavy Chain Nucleotide Sequence 3: [C225v7 (SEQ ID NO: 425)] (SEQ ID NO: 425)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaccaragcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga] 3954-1204-C225v7 Activatable Antibody Heavy Chain Amino Acid Sequence 3: [C225v7 (SEQ ID NO: 427)] (SEQ ID NO: 427)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*]3954-1204-C225v7 Activatable Antibody Light Chain Nucleotide Sequence 3:[Mask (SEQ ID NO: 304)][Linker 1 (SEQ ID NO: 305)][1204Substrate (SEQ ID NO: 306)][Linker 2 (SEQ ID NO: 307)][C225 (SEQ ID NO: 308)] (SEQ ID NO: 413)[tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac][ ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataatcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v7 Activatable Antibody Light Chain Amino Acid Sequence 3:[Mask (SEQ ID NO: 113)][Linker 1 (SEQ ID NO: 293)][1204Substrate (SEQ ID NO: 266)][Linker 2 (SEQ ID NO: 294)][C225 (SEQ ID NO: 310)] (SEQ ID NO: 414) [CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][LSGRSDNH ][

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Exemplary conjugated antibodies and/or activatable antibodies of theinvention include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the antibody referred to herein as the“AV1”antibody, which binds interleukin-6 receptor (IL-6R). The amino acidsequences for the Av1 heavy chain and the Av1 light chain are shownbelow in SEQ ID NO: 330 and SEQ ID NO: 331, respectively.

Av1 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 330)QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAv1 Antibody Light Chain Amino Acid Sequence: (SEQ ID NO: 331)DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Exemplary conjugated antibodies and/or activatable antibodies of theinvention include, for example, antibodies that bind interleukin 6receptor (IL-6R) and that include a heavy chain and a light chain thatare, or are derived from, the Av1 antibody and a masking moiety.Exemplary conjugated antibodies and/or activatable antibodies of theinvention include an amino acid sequence attached to the N-terminus ofthe AV1 light chain. These N-terminal masking moiety amino acidsequences include, for example, YGSCSWNYVHIFMDC (SEQ ID NO: 332);QGDFDIPFPAHWVPIT (SEQ ID NO: 333); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 334);QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 335); QGQSGQGDFDIPFPAHWVPIT (SEQ IDNO: 336); or QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 337). It is also to beappreciated that such amino acid sequences can be attached to theN-terminus of the AV1 heavy chain or to the C-terminus of the AV1 heavyor light chain.

Exemplary activatable antibodies of the invention include, for example,antibodies that bind a Jagged target, e.g., Jagged-1, Jagged-2 and/orboth Jagged-1 and Jagged-2, and that include a combination of a variableheavy chain region and a variable light chain region that are, or arederived from, the variable heavy chain and variable light chainsequences shown below.

Variable Light Chain Amino Sequence Lc4 (SEQ ID NO: 338)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc4 (SEQ ID NO: 339)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc5 (SEQ ID NO: 340)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc5 (SEQ ID NO: 341)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc7 (SEQ ID NO: 342)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc7 (SEQ ID NO: 343)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc8 (SEQ ID NO: 344)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc8 (SEQ ID NO: 345)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc13 (SEQ ID NO: 346)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc13 (SEQ ID NO: 347)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc16 (SEQ ID NO: 348)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc16 (SEQ ID NO: 349)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc19 (SEQ ID NO: 350)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc19 (SEQ ID NO: 351)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc21 (SEQ ID NO: 352)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc21 (SEQ ID NO: 353)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc24 (SEQ ID NO: 354)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc24 (SEQ ID NO: 355)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc26 (SEQ ID NO: 356)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc26 (SEQ ID NO: 357)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc27 (SEQ ID NO: 358)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc27 (SEQ ID NO: 359)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc28 (SEQ ID NO: 360)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc28 (SEQ ID NO: 361)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc30 (SEQ ID NO: 362)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc30 (SEQ ID NO: 363)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc31 (SEQ ID NO: 364)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc31 (SEQ ID NO: 365)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc32 (SEQ ID NO: 366)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc32 (SEQ ID NO: 367)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc37 (SEQ ID NO: 368)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc37 (SEQ ID NO: 369)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc39 (SEQ ID NO: 370)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc39 (SEQ ID NO: 371)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc40 (SEQ ID NO: 372)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRHeavy Chain Amino Sequence Hc40 (SEQ ID NO: 373)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc47 (SEQ ID NO: 374)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKRVariable Heavy Chain Amino Sequence Hc47 (SEQ ID NO: 375)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable 4B2 Light Chain (SEQ ID NO: 376)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR Variable 4B2 Heavy Chain(SEQ ID NO: 377)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable 4D11 Light Chain (SEQ ID NO: 378)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR Variable 4D11 Heavy Chain(SEQ ID NO: 379)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable 4E7 Light Chain (SEQ ID NO: 380)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR Variable 4E7 Heavy Chain(SEQ ID NO: 381)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable 4E11 Light Chain (SEQ ID NO: 382)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR Variable 4E11 Heavy Chain(SEQ ID NO: 383)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSVariable 6B7 Light Chain (SEQ ID NO: 384)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6B7 Heavy Chain(SEQ ID NO: 385)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSSVariable 6F8 Light Chain (SEQ ID NO: 386)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6F8 Heavy Chain(SEQ ID NO: 387)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS

Exemplary activatable antibodies of the invention include, for example,antibodies that bind a Jagged target, e.g., Jagged-1, Jagged-2 and/orboth Jagged-1 and Jagged-2, and that include a combination of a heavychain region and a light chain region that are, or are derived from, theheavy chain and light chain sequences shown below.

4D11 Light Chain sequence: (SEQ ID NO: 388)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC4D11 Heavy Chain sequence: (SEQ ID NO: 389)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK4D11v2 Heavy Chain sequence (SEQ ID NO: 390)EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK4D11v2 Light Chain Sequence (SEQ ID NO: 391)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

An exemplary activatable anti-Jagged antibody of the invention is theactivatable antibody referred to herein as the 5342-1204-4D11activatable antibody, which binds human Jagged 1 and human Jagged 2 whenthe activatable antibody is in an activated state. Two sequences of the5342-1204-4D11 activatable anti-Jagged antibody are shown below,Sequence 1 is the sequence of a version of the 5342-1204-4D11activatable anti-Jagged antibody that includes a spacer peptide, andSequence 2 is the sequence of the 5342-1204-4D11 activatable anti-Jaggedantibody without the spacer sequence:

5342-1204-4D11 Activatable Antibody Heavy Chain Nucleotide Sequence 1:(SEQ ID NO: 415)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA5342-1204-4D11 Activatable Antibody Heavy Chain Amino Acid Sequence 1:(SEQ ID NO: 416)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K]5342-1204-4D11 Activatable Antibody Light Chain Nucleotide Sequence 1:(SEQ ID NO: 417)CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGA GAGTGT5342-1204-4D11 Activatable Antibody Light Chain Amino Acid Sequence 1:(SEQ ID NO: 418)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC5342-1204-4D11 Activatable Antibody Heavy Chain Nucleotide Sequence 2:(SEQ ID NO: 415)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA5342-1204-4D11 Activatable Antibody Heavy Chain Amino Acid Sequence 2:(SEQ ID NO: 416)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K]5342-1204-4D11 Activatable Antibody Light Chain Nucleotide Sequence 2:(SEQ ID NO: 419)TGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT5342-1204-4D11 Activatable Antibody Light Chain Amino Acid Sequence 2:(SEQ ID NO: 420)CNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

When an activatable antibody is cleaved by a protease, i.e., when theactivatable antibody is in an active or cleaved state, the activatedantibody will retain only a portion of the amino acid sequence of theactivatable antibody in an inactive or uncleaved state. The sequence ofthe activatable antibody in an active or cleaved state will varydepending on which protease cleaves the substrate (CM), as differentproteases can have different recognition sites.

For example, when an activatable anti-EGFR antibody is cleaved by aprotease, i.e., when the activatable anti-EGFR antibody is in an activeor cleaved state, the activated anti-EGFR antibody will retain only aportion of the amino acid sequence of the activatable antibody in aninactive or uncleaved state. The sequence of the activatable anti-EGFRantibody in an active or cleaved state will vary depending on whichprotease cleaves the substrate (CM), as different proteases can havedifferent recognition sites.

Examples of various N-terminal sequences of the light chain of activatedversions 3954-1204-C225v5 anti-EGFR activatable antibody, where theactivatable antibody has been activated by various proteases is shownbelow in Table 5, where the N-terminal portion of the uncleaved sequence(−) is SEQ ID NO: 392, the N-terminal portion of thematriptase-activated sequence is SEQ ID NO: 393, the N-terminal portionof the uPA activated sequence is SEQ ID NO: 393, and the N-terminalportion of the legumain activated sequence is SEQ ID NO: 394. Theannotated sequences show the residues that compose the masking moiety(MM), the first linker peptide (LP1), the substrate (CM), the secondlinker peptide (LP2), and the N-terminal residue of the C225v5 antibody(i.e., the N-terminal Q residue of the C225v5 AB).

TABLE 5

In some embodiments, the activatable antibodies described herein alsoinclude an agent conjugated to the activatable antibody. In someembodiments, the conjugated agent is a therapeutic agent, such as anantineoplastic agent. In such embodiments, the agent is conjugated to acarbohydrate moiety of the activatable antibody, for example, where thecarbohydrate moiety is located outside the antigen-binding region of theantibody or antigen-binding fragment in the activatable antibody. Insome embodiments, the agent is conjugated to a sulfhydryl group of theantibody or antigen-binding fragment in the activatable antibody. Insome embodiments, the agent is conjugated to an amino group of theantibody or antigen-binding fragment of the activatable antibody. Insome embodiments the agent is conjugated to a carboxylic acid group ofthe antibody or antigen-binding fragment of the activatable antibody.

In some embodiments, the agent is a cytotoxic agent such as a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Suitable cytotoxic agents include, for example, any ofthe cytotoxic agents listed in Table 4. In some embodiments, thecytotoxic agent is a dolastatin or a derivative thereof (e.g. auristatinE, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the cytotoxic agentis monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD). Insome embodiments, the agent is an agent selected from the group listedin Table 4. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is monomethyl auristatin D (MMAD). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof. In some embodiments, theagent is a pyrrolobenzodiazepine.

In some embodiments, the conjugated activatable antibody can be modifiedfor site-specific conjugation through modified amino acid sequencesinserted or otherwise included in the activatable antibody sequence.These modified amino acid sequences are designed to allow for controlledplacement and/or dosage of the conjugated agent within a conjugatedactivatable antibody. For example, the activatable antibody can beengineered to include cysteine substitutions at positions on light andheavy chains that provide reactive thiol groups and do not negativelyimpact protein folding and assembly, nor alter antigen binding. In someembodiments, the activatable antibody can be engineered to include orotherwise introduce one or more non-natural amino acid residues withinthe activatable antibody to provide suitable sites for conjugation. Insome embodiments, the activatable antibody can be engineered to includeor otherwise introduce enzymatically activatable peptide sequenceswithin the activatable antibody sequence.

In some embodiments, the agent is a detectable moiety such as, forexample, a label or other marker. For example, the agent is or includesa radiolabeled amino acid, one or more biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), one or more radioisotopes or radionuclides, oneor more fluorescent labels, one or more enzymatic labels, and/or one ormore chemiluminescent agents. In some embodiments, detectable moietiesare attached by spacer molecules.

Enzymatically active toxins and antigen-binding fragments thereof thatcan be used include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Table 4 lists some of the exemplary pharmaceutical agents but in no wayis meant to be an exhaustive list.

TABLE 4 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTSAuristatins Auristatin E Monomethyl auristatin E (MMAE) Monomethylauristatin D (MMAD) Desmethyl auristatin E (DMAE) Auristatin FMonomethyl auristatin F (MMAF) Desmethyl auristatin F (DMAF) Auristatinderivatives, e.g., amides thereof Auristatin tyramine Auristatinquinoline Dolastatins Dolostatin 10 Dolastatin derivatives Dolastatin 16DmJ Dolastatin 16 Dpv Maytansinoids, e.g. DM-1; DM-4 Maytansinoidderivatives Duocarmycin Duocarmycin derivatives Alpha-amanitinAnthracyclines Doxorubicin Daunorubicin Bryostatins CamptothecinCamptothecin derivatives 7-substituted Camptothecin 10, 11-Difluoromethylenedioxycamptothecin Combretastatins DebromoaplysiatoxinKahalalide-F Discodermolide Ecteinascidins ANTIVIRALS Acyclovir Vira ASymmetrel ANTIFUNGALS Nystatin ADDITIONAL ANTI-NEOPLASTICS AdriamycinCerubidine Bleomycin Alkeran Velban Oncovin Fluorouracil MethotrexateThiotepa Bisantrene Novantrone Thioguanine Procarabizine CytarabineANTI-BACTERIALS Aminoglycosides Streptomycin Neomycin Kanamycin AmikacinGentamicin Tobramycin Streptomycin B Spectinomycin AmpicillinSulfanilamide Polymyxin Chloramphenicol Turbostatin PhenstatinsHydroxyphenstatin Spongistatin 5 Spongistatin 7 Halistatin 1 Halistatin2 Halistatin 3 Modified Bryostatins Halocomstatins Pyrrolobenzimidazoles(PBI) Cibrostatin6 Doxaliform Anthracyclins analogues Cemadotin analogue(CemCH2-SH) Pseudomonas toxin A (PE38) variant Pseudomonas toxin A(ZZ-PE38) variant ZJ-101 OSW-1 4-Nitrobenzyloxycarbonyl Derivatives ofO6-Benzylguanine Topoisomerase inhibitors Hemiasterlin CephalotaxineHomoharringtonine Pyrrolobenzodiazepine dimers (PBDs) Functionalizedpyrrolobenzodiazepenes Calicheamicins Podophyllotoxins Taxanes Vincaalkaloids CONJUGATABLE DETECTION REAGENTS Fluorescein and derivativesthereof Fluorescein isothiocyanate (FITC) RADIOPHARMACEUTICALS ¹²⁵I ¹³¹I⁸⁹Zr ¹¹¹In ¹²³I ¹³¹I ⁹⁹mTc ²⁰¹Tl ¹³³Xe ¹¹C ⁶²Cu ¹⁸F ⁶⁸Ga ¹³N ¹⁵O ³⁸K⁸²Rb ⁹⁹mTc (Technetium) HEAVY METALS Barium Gold PlatinumANTI-MYCOPLASMALS Tylosine Spectinomycin

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The binding is, insome embodiments, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Suitable linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Suitablelinkers include: (i) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, e.g., cleavable ornon-cleavable, or the two or more linkers are different, e.g., at leastone cleavable and at least one non-cleavable.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; in some embodiments ≦100 nM and in some embodiments ≦10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type that occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present invention is said to specifically bind to EGFR,when the equilibrium binding constant (K_(d)) is ≦1 μM, in someembodiments ≦100 nM, in some embodiments ≦10 nM, and in some embodiments≦100 pM to about 1 pM, as measured by assays such as radioligand bindingassays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide that it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of rabbit or murineproteins, (3) is expressed by a cell from a different species, or (4)does not occur in nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. For example, oligonucleotides are 10 to 60bases in length and in some embodiments, 12, 13, 14, 15, 16, 17, 18, 19,or 20 to 40 bases in length. Oligonucleotides are usually singlestranded, e.g., for probes, although oligonucleotides may be doublestranded, e.g., for use in the construction of a gene mutant.Oligonucleotides of the invention are either sense or antisenseoligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, in some embodiments, at least 90 percent sequenceidentity, in some embodiments, at least 95 percent sequence identity,and in some embodiments, at least 99 percent sequence identity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, in some embodiments, atleast 80%, 90%, 95%, and in some embodiments, 99%. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic amino acids areaspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site. Whether an amino acid change resultsin a functional peptide can readily be determined by assaying thespecific activity of the polypeptide derivative. Assays are described indetail herein. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Suitable amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. In some embodiments,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

Suitable amino acid substitutions are those that: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(e.g., conservative amino acid substitutions) may be made in thenaturally-occurring sequence (e.g., in the portion of the polypeptideoutside the domain(s) forming intermolecular contacts. A conservativeamino acid substitution should not substantially change the structuralcharacteristics of the parent sequence (e.g., a replacement amino acidshould not tend to break a helix that occurs in the parent sequence, ordisrupt other types of secondary structure that characterizes the parentsequence). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples (Creighton, Ed., W. H. Freeman and Company, New York (1984));Introduction to Protein Structure (C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, in some embodiments, at least 14 amino acids long, in someembodiments, at least 20 amino acids long, usually at least 50 aminoacids long, and in some embodiments, at least 70 amino acids long. Theterm “analog” as used herein refers to polypeptides that are comprisedof a segment of at least 25 amino acids that has substantial identity toa portion of a deduced amino acid sequence and that has specific bindingto EGFR, under suitable binding conditions. Typically, polypeptideanalogs comprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, in some embodiments, atleast 50 amino acids long or longer, and can often be as long as afull-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and in someembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, in some embodiments, more than about 85%, 90%, 95%, and99%. In some embodiments, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects. The termspatient and subject are used interchangeably herein. In someembodiments, the subject is a mammal, such as a human, non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a human. Insome embodiments, the subject is a companion animal. In someembodiments, the subject is an animal in the care of a veterinarian.

Use of Antibodies that Bind Activatable Antibodies

It will be appreciated that administration of antibodies andantigen-binding fragments thereof in accordance with the invention willbe administered with suitable carriers, excipients, and other agentsthat are incorporated into formulations to provide improved transfer,delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences (15th ed, Mack PublishingCompany, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour,therein. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as Lipofectin™), DNA conjugates, anhydrousabsorption pastes, oil-in-water and water-in-oil emulsions, emulsionscarbowax (polyethylene glycols of various molecular weights), semi-solidgels, and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Antibodies and antigen-binding fragments thereof can be administered inthe form of compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) MackPub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,Possibilities, Limitations, And Trends, Harwood Academic Publishers,Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest fragment thatspecifically binds to the target activatable antibody and/or conjugatedactivatable antibody is used in some embodiments. For example, basedupon the variable-region sequences of an antibody, peptide molecules canbe designed that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl.Acad. Sci. USA, 90: 7889-7893 (1993)).

In some embodiments, the antibody contains a detectable label. An intactantibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)₂) is used.The term “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently-labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently-labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component ofblood including blood serum, blood plasma, or lymph. That is, thedetection method of the invention can be used to detect an analyte mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of an analyte mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of an analyte protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations,immunochemical staining, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Diagnostic Formulations

Antibodies and/or fragments of the disclosure are also useful in thedetection of activatable antibodies and/or conjugated activatableantibodies in patient samples and accordingly are useful as diagnostics.For example, the antibodies and antigen-binding fragments thereof thatbind activatable antibodies and/or conjugated activatable antibodies areused in in vitro assays, e.g., ELISA, to detect activatable antibodiesand/or conjugated activated antibodies levels in a patient sample. Insome embodiments, the antibodies and antigen-binding fragments thereofthat bind activatable antibodies and/or conjugated activatableantibodies are used in in vitro assays, e.g., ELISA, to detect the totallevel (activated and non-activated) of activatable antibodies and/orconjugated activated antibodies and/or the intact level (non-activated)activatable antibodies and/or conjugated activated antibodies as shownin the Examples provided herein.

In one embodiment, an antibody or fragment of the disclosure isimmobilized on a solid support (e.g., the well(s) of a microtiterplate). The immobilized antibody and/or fragment serves as a captureantibody for any activatable antibody and/or conjugated activatableantibody that may be present in a test sample. Prior to contacting theimmobilized antibody with a sample, the solid support is rinsed andtreated with a blocking agent such as milk protein or albumin to preventnonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of activatable antibody and/or conjugated activatableantibody in the test sample is determined by comparison with a standardcurve developed from the standard samples.

An antibody and/or fragment of the disclosure can also be used indiagnostic and/or imaging methods. In some embodiments, such methods arein vitro methods. In some embodiments, such methods are in vivo methods.In some embodiments, such methods are in situ methods. In someembodiments, such methods are ex vivo methods.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Generation of Antibodies that Bind ActivatableAntibodies

Exemplary antibodies that bind activatable antibodies and/or conjugatedactivatable antibodies include the antibodies referred to herein as 41(also referred to herein as 41-2 and/or clone 41), 58 (also referred toherein as 58-1 and/or clone 58), 72 (also referred to herein as 72-3and/or clone 72), and 85 (also referred to herein as 85-1 and/or clone85) and antigen-binding fragments thereof. These antibodies weregenerated using the following peptide antigen (SEQ ID NO: 395):QGQSGQCISPRGCPDGPYVMYGSSGGSGGSK; which includes a disulfide bridgebetween C7 and C16. This 33 amino acid peptide represents the sequenceof the masking moiety (MM) and the first linker peptide (LP1) of theactivatable anti-EGFR antibody referred to herein as 3954-1204-c225v5.This peptide antigen was conjugated to two different carrier proteinsfor immunization: immunization: (i) 5 mg to Keyhole Limpet Hemocyanin(KLH) or (ii) 3 mg to ovalbumin (OVA). Selected rabbits were alsoimmunized with the full length version of the 3954-1204-c225v5 anti-EGFRactivatable antibody.

The rabbits were immunized using the following procedure. Four threemonth old New Zealand white rabbits were immunized using a customizedprotocol of 5 or 6 injections. At the time of each injection, theantigen aliquot was thawed and combined with Complete Freund's Adjuvant(CFA) (for the first injection) or with incomplete Freund's Adjuvant(IFA) for the subsequent injections. The injection route wassubcutaneous (SC).

Two rabbits were immunized with the conjugated peptide and two otherrabbits were immunized with the conjugated peptide and the full lengthversion of the 3954-1204-c225v5 anti-EGFR activatable antibody.

Serum titer against the free peptide or the activatable anti-EGFRantibody as well as counter screen antigen (human IgG) was evaluatedusing test bleeds using a standard ELISA procedure. The resultsindicated that all 4 rabbits had comparable titers against therespective immunogen and were ready for splenectomy.

Splenocytes from the immunized rabbits were isolated using the followingmethod. Three rabbits were selected for final intravenous boost andsplenectomy. Splenocytes from a rabbit immunized with the3954-1204-c225v5 and the peptide antigen and a rabbit immunized with thepeptide only were used for monoclonal development. The final intravenousboost was performed with OVA-conjugated peptide (2 rabbits immunizedwith peptide only) or a mixture of OVA conjugated peptide and3954-1204-c225v5 (one rabbit immunized with peptide and3954-1204-c225v5).

Rabbit monoclonal antibodies were generated as follows: Lymphocytes fromrabbits E5251 and E5253 were used for hybridoma fusion with partnercells 240E-W2 and plated on forty 96-wells plates (400 millionlymphocytes per rabbit). The plates were kept in tissue cultureincubators under standard conditions.

Clones 41, 58, 72, 85 were selected for subcloning and characterization.

Molecular cloning was performed using the following method. mRNA fromhybridoma cells was isolated using a TuboCapture Kit (Qiagen: Catalog#72232) following the manufacturer's instruction and reverse transcribedinto cDNA using oligo-dT primer. The variable region of heavy chain (VH)was PCR amplified using primers OYZ64-2 and OYZvh3. The entire lightchain (LC) was PCR amplified using primers OYZ62 and OYZ71. The VHregion of PCR fragments was digested using restriction enzyme HindIIIand KpnI. The LC PCR fragments were digested using HindIII and NotI. Alldigested products were purified using Qiagen QIAquick PCR PurificationKit (catalog #28014). After purification, the VH or LC fragment,respectively, was ligated into the corresponding heavy or light chainproprietary expression vectors and transformed into competent cells DH5α(MC Lab, catalog #DA-100). The transformed colonies were picked andinserts were confirmed (by expected size: approximately 440 bp for VHand 740 bp for LC) using the corresponding restriction enzymes. Plasmidswith inserts of the expected sizes were sequenced using the TT5 primer.The sequences for VH or LC regions are provided herein. The light chainand heavy chain encoding nucleic acid molecules were co-transfected into293 cells in 6-well plates. The supernatants were collected five dayspost transfection and tested against corresponding antigen. In addition,the IgG concentration was measured by ELISA.

In addition, the entire light chain and heavy chain fragments wereexcised from the corresponding vector with HindIII and NotI andsubsequently purified using Qiagen QIAquick PCR Purification Kit(catalog #28014).

Example 2 Binding Specificity of Antibodies that Bind Anti-EGFRActivatable Antibodies

Microsorp (Nunc) 96-well plate(s) were coated with 50 μl/well of 1 μg/ml(i) cetuximab, (ii) parental antibody C225v5 (a variant of cetuximab,the sequence of which is provided herein), (iii) a masked anti-EGFRantibody, referred to herein as 3954-NSUB-c225v5, which contains thenoncleavable sequence GSSGGSGGSGGSGGGSGGGSGGS (SEQ ID NO: 396) betweenthe mask and the light chain of the anti-EGFR antibody C225v5; (iv) anactivatable anti-EGFR antibody referred to herein as 3954-1204-c225v5;(v) the 3954-1204-c225v5 activatable anti-EGFR antibody activated withuPA, or (vi) the anti-VEGF-A antibody bevacizumab; each of theantibodies in PBS overnight at 4° C. The plates were washed withPBS/0.05% Tween 20 (wash butter) and then blocked with PBS/1% BSA for 1hour at room temperature. The blocking buffer was removed, and theanti-AA antibodies 41, 58, 72 or 85, i.e., antibodies that bindactivatable antibodies and/or conjugated activatable antibodies, wereadded and incubated for 1 hour. The plates were washed with wash bufferand incubated with 50 μl of 1 μg/ml of horseradish peroxidase conjugateddonkey anti-rabbit IgG antibody for 1 hour. The plates were washed withwash buffer and 100 μl/well of TMB substrate was added. The reaction wasstopped by the addition of 100 μl 1M HCl and the OD 450 nm was measured.

As shown in FIG. 1, each anti-AA antibody demonstrated a different butoverlapping specificity. Clone 41 bound to all antibodies tested(cetuximab, C225v5, intact 3954-1204-c225v5, 3954-NSUB-c225v5), with theexception of bevacizumab. Clone 58 bound with greater specificity to theintact 3954-1204-c225v5 but also bound weakly to 3954-NSUB-c225v5. Clone72 bound with greater specificity to 3954-1204-c225v5 but alsorecognized 3954-NSUB-c225v5 well. Clone 72 also demonstrated a very weakbinding to the uPA activated 3954-1204-c225v5. Clone 85 bound equallywell to 3954-1204-c225v5 and 3954-NSUB-c225v5 but did not bind to anyother antibody tested.

Example 3 Quantification of Total (Activated and Non-Activated)Activatable Antibodies and Intact (Non-Activated) Activatable Antibodies

The antibodies and antigen-binding fragments thereof that bindactivatable antibody and/or conjugated activatable antibodies, i.e.,anti-AA antibodies of the disclosure, were used to develop assays tomeasure the concentration of total (activated and non-activated) andintact (non-activated) activatable antibodies, using the3954-1204-c225v5 anti-EGFR activatable antibody as an example.

Microsorp (Nunc) 96-well plate(s) were coated with 50 μl/well, 1 μg/mlanti-AA clone 41 in PBS overnight at 4 C. The plates were washed withPBS/0.05% Tween 20 (wash buffer) and blocked with PBS/1% BSA for 1 hourat room temperature. The blocking buffer was removed and 50 μl/well ofsample were added and incubated for 1 hour. Intact or partially (˜50%)activated 3954-1204-c225v5 in human plasma was used as each sample. Theplates were washed with wash buffer and incubated with 50 μl (e.g., at 2μg/ml) biotinylated anti-AA antibodies (clones 58 or 72) or biotinylatedgoat anti-human IgG for 1 hour at room temperature. The plates werewashed with wash buffer and incubated for 30 min with 1 μg/ml horseradish peroxidase conjugated streptavidin. The plates were washed withwash buffer and the binding was measured using a colorimetric substrateby incubating 100 μl/well of TMB substrate. The reaction was stopped bythe addition of 100 μl 1M HCl and the OD 450 nm was measured.

When the biotinylated goat anti-human IgG was used, the detection of3954-1204-c225v5 gave a high signal for the intact 3954-1204-c225v5 andthe partially activated 3954-1204-c225v5. However, when the biotinylatedclones 58 or the clone 72 were used, the detection of the partiallyactivated 3954-1204-c225v5 gave a signal approximately 50% lowerrelative to the signal given by the detection of the intact3954-1204-c225v5. These data showed that a combination of theseantibodies can be used to develop an assay to measure the concentrationof total and intact 3954-1204-c225v5 in human plasma. These assays are,therefore, useful in methods to measure concentrations of total andintact 3954-1204-c225v5, for example in treated subjects.

Example 4 Use of Antibodies that Bind Activatable Antibodies to DetectActivatable Antibodies in Tumor Samples

The example provided herein demonstrates the detection of cetuximab,activatable anti-EGFR antibody (3954-1204-c225v5), and masked anti-EGFRantibody (3954-NSUB-c225v5) by immunofluorescence using the anti-AAantibody clone 41. Nude mice bearing a subcutaneous cell line xenografttumor (H292 non-small cell lung cancer; NSCLC) or patient derived tumor(LXFA NSCLC) were injected intraperitoneally with cetuximab,3954-1204-c225v5, 3954-NSUB-c225v5, or PBS. After the injection (72 h)the tumors were excised and embedded in OCT and frozen. The tumors werecryosectioned and immunofluorescence was performed using clone 41 and aFITC-conjugated anti-rabbit IgG.

FIG. 3 demonstrates that immunofluorescent detection with clone 41revealed a strong signal for tumors from mice treated with cetuximab and3954-1204-c225v5 but a lower signal for tumors from mice injected with3954-NSUB-c225v5. No fluorescent signal was detected in the tumorsresected from mice injected with PBS. These results suggest that theanti-AA antibody clone 41 is useful in methods to detect cetuximab,3954-1204-c225v5, or 3954-NSUB-c225v5, for example in tissues fromtreated animal or human subjects.

Example 5 Generation of Additional Antibodies that Bind ActivatableAntibodies

In addition to the antibodies descried in Example 1, exemplaryantibodies that bind activatable antibodies and/or conjugatedactivatable antibodies include the antibodies referred to herein as 10(also referred to herein as 10-10 and/or clone 10), 8 (also referred toherein as 8-8 and/or clone 8), 53 (also referred to herein as 53-1and/or clone 3), 7 (also referred to herein as 7-11 and/or clone 7), 36(also referred to herein as 36-3 and/or clone 36), 52 (also referred toherein as 52-10 and/or clone 52), and 27 (also referred to herein as27-4 and/or clone 27), and antigen-binding fragments thereof. Theseantibodies were generated using the following peptide antigen (SEQ IDNO: 397): QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSK, which includesa disulfide bridge between C7 and C16. This 45 amino acid peptiderepresents the sequence of the masking moiety (MM) and the first linkerpeptide (LP1) of the activatable anti-Jagged-1/-2 antibody referred toherein as 5342-1204-4D11, which comprises the heavy chain sequence ofSEQ ID NO: 416 and the light chain sequence of SEQ ID NO: 420. Thispeptide antigen was conjugated to two different carrier proteins forimmunization: immunization: (i) 2 mg to Keyhole Limpet Hemocyanin (KLH)or (ii) 2 mg to ovalbumin (OVA). Selected rabbits were also immunizedwith the full length version of the 5342-1204-4D11 anti-Jagged-1/-2activatable antibody.

The rabbits were immunized using the following procedure. Two threemonth old New Zealand white rabbits were immunized using a customizedprotocol of 6 injections. The rabbits were alternatively injected withthe conjugated peptide and the full length version of the 5342-1204-4D11anti-Jagged-1/-2 activatable antibody at a two weeks interval schedule.At the time of each injection, the antigen aliquot was thawed andcombined with Complete Freund's Adjuvant (CFA) (for the first injection)or with incomplete Freund's Adjuvant (IFA) for the subsequentinjections. The injection route was subcutaneous (SC).

Serum titer against the free peptide or the activatable anti-Jagged-1/-2antibody as well as counter screen antigen (human IgG) was evaluatedusing test bleeds using a standard ELISA procedure. The resultsindicated that both rabbits had comparable titers against the respectiveimmunogen and were ready for splenectomy.

Splenocytes from the immunized rabbits were isolated using the followingmethod. The selected rabbit received a final intravenous boost andsplenectomy. The final intravenous boost was performed withOVA-conjugated peptide.

Rabbit monoclonal antibodies were generated as follows: Lymphocytes fromrabbits #359 were used for hybridoma fusion with partner cells 240E-W2and plated on forty 96-wells plates (400 million lymphocytes perrabbit). The plates were kept in tissue culture incubators understandard conditions.

Clones 10, 8, 53, 7, 36, 52, and 27 were selected for subcloning andcharacterization. The following final subclones were selected, 10-10,8-8, 53-1, 7-11, 36-3, 52-10, and 27-4.

Example 6 Binding Specificity of Antibodies that Bind Anti-Jagged-1/-2Activatable Antibodies

Microsorp (Nunc) 96-well plate(s) were coated with 50 μl/well of 1 μg/ml(i) parental antibody 4D11, (ii) an activatable anti-Jagged-1/-2antibody referred to herein as 5342-1204-4D11, which comprises the heavychain sequence of SEQ ID NO: 416 and the light chain sequence of SEQ IDNO: 420; (iii) a masked anti-Jagged-1/-2 antibody, referred to herein as5342-NSUB-4D11, which comprises the heavy chain sequence of SEQ ID NO:416 and contains the noncleavable sequence GSSGGSGGSGGSGGGSGGGSGGS (SEQID NO: 396) between the mask and the light chain of the anti-Jagged-1/-2antibody 4D11 as shown below in SEQ ID NO: 421; (iv) the 5342-1203-4D11activatable anti-Jagged-1/-2 antibody, which comprises the heavy chainsequence of SEQ ID NO: 416 and the light chain sequence of SEQ ID NO:422 shown below; (v) an activatable anti-Jagged-1/-2 antibody referredto herein as 5342-PLGL-4D11, which comprises the heavy chain sequence ofSEQ ID NO: 416 and the light chain sequence of SEQ ID NO: 423 shownbelow; (vi) an activatable anti-EGFR antibody referred to herein as3954-1204-C225v5, which comprises the heavy chain sequence of SEQ ID NO:301 and the light chain sequence of SEQ ID NO: 410; (vii) an activatableanti-Jagged-1/-2 antibody referred to herein as 5872-1204-4D11, whichcomprises the heavy chain sequence of SEQ ID NO: 416 and the light chainsequence of SEQ ID NO: 424 shown below, (viii) pooled human IgG(Gammaguard); or (ix) the 5342-1204-4D11 activatable anti-Jagged-1/-2antibody activated with uPA. Each of the antibodies was incubated in PBSovernight at 4° C.

5342-NSUB-4D11 Activatable Antibody Light Chain Amino Acid Sequence:(SEQ ID NO: 421) CNIWLVGGDCRGWQGGSSGGSGGSGGGSSGGSGGSGGSGGGSGGGSGGSGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC5342-1203-4D11 Activatable Antibody Light Chain Amino Acid Sequence:(SEQ ID NO: 422) CNIWLVGGDCRGWQGGSSGGSGGSGGTGRGPSWVGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC5342-PLGL-4D11 Activatable Antibody Light Chain Amino Acid Sequence:(SEQ ID NO: 423) CNIWLVGGDCRGWQGGSSGGSGGSGGSGGGSPLGLGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC5872-1204-4D11 Activatable Antibody Light Chain Amino Acid Sequence:(SEQ ID NO: 424) GCNIWLNGGDCRGWVDPLQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

The plates were washed with PBS/0.05% Tween 20 (wash buffer) and thenblocked with PBS/1% BSA for 1 hour at room temperature. The blockingbuffer was removed, and the anti-AA antibodies 10, 8, 53, 7, 36, 52, and27, i.e., antibodies that bind activatable antibodies and/or conjugatedactivatable antibodies, were added and incubated for 1 hour. The plateswere washed with wash buffer and incubated with 50 μl of 1 μg/ml ofhorseradish peroxidase conjugated donkey anti-rabbit IgG antibody for 1hour. The plates were washed with wash buffer and 100 μl/well of TMBsubstrate was added. The reaction was stopped by the addition of 100 μl1M HCl and the OD 450 nm was measured.

As shown in Table 6, each anti-AA antibody demonstrated a different butoverlapping specificity:

TABLE 6 Specificity of Anti-Activatable Anti-Jagged-1/-2 Antibodies10-10 8-8 53-1 7-11 36-3 52-10 27-4 4D11 − − − − + ++ +++ 5342-1204-4D11+++ ++ ++ +++ +++ ++ +++ 5342-NSUB-4D11 +++ ++ ++ +++ + ++ +++5342-1203-4D11 +++ ++ ++ +++ + ++ +++ 5342-PLGL-4D11 +++ ++ ++ +++ + +++++ 3954-1204-C225v5 − − − +++ +++ − − 5872-1204-4D11 − ++ ++ +++ +++ +++++ IvIg − − − − − − − active 5342-1204-4D11 − − ++ + ++ N/A N/A

Clone 10 bound only to the activatable anti-Jagged-1/-2 antibodiesbearing the 5342 mask tested (5342-1204-4D11, 5342-NSUB-4D11,5342-1203-4D11, 5342-PLGL-4D11). Clone 8 bound only to the intactactivatable anti-Jagged-1/-2 antibodies tested (5342-1204-4D11,5342-NSUB-4D11, 5342-1203-4D11, 5342-PLGL-4D11, 5872-1204-4D11). Clone53 bound to all the activatable anti-Jagged-1/-2 antibodies tested(5342-1204-4D11, 5342-NSUB-4D11, 5342-1203-4D11, 5342-PLGL-4D11,5872-1204-4D11, activated 5342-1204-4D11). Clone 7 bound to all theactivatable antibodies tested (5342-1204-4D11, 5342-NSUB-4D11,5342-1203-4D11, 5342-PLGL-4D11, 3954-1204-C225v5, 5872-1204-4D11,activated 5342-1204-4D11) with a greater binding to the intactactivatable antibodies compared to the activated one. Clone 36 bound toall the antibodies tested at the exception of MG and demonstrated agreater binding to the activatable antibodies bearing the 1204substrate. Clone 52 and 27 showed a similar specificity and bound to theanti-Jagged-1/-2 antibody and all the activatable anti-Jagged-1/-2antibodies tested (5342-1204-4D11, 5342-NSUB-4D11, 5342-1203-4D11,5342-PLGL-4D11, 5872-1204-4D11).

Example 7 Effect of Human Serum on the Binding of Anti-AA to theActivatable Anti-Jagged-1/-2 Antibody 5342-1204-4D11

The effect of the presence of human serum on the binding of antibodiesto the activatable anti-Jagged-1/-2 5342-1204-4D11 was tested by ELISA.

Microsorp (Nunc) 96-well plate(s) were coated with 50 μl/well, 1 μg/ml5342-1204-4D11 in PBS overnight at 4° C. The plates were washed withPBS/0.05% Tween 20 (wash buffer) and blocked with PBS/1% BSA for 1 hourat room temperature. The blocking buffer was removed and the anti-AAantibodies 10, 8, 53, 7, 36, 52, and 27, i.e., antibodies that bindactivatable antibodies and/or conjugated activatable antibodies, wereadded and incubated for 1 hour in the presence of human serum (0%-50%).The plates were washed with wash buffer and incubated with 50 μl of 1μg/ml of horseradish peroxidase conjugated donkey anti-rabbit IgGantibody for 1 hour. The plates were washed with wash buffer and 100μl/well of TMB substrate was added. The reaction was stopped by theaddition of 100 μl 1M HCl and the OD 450 nm was measured.

As shown in FIG. 4, the binding of the anti-AA 10 and 36 was not alteredby the presence of human serum. However in the presence of increasingconcentrations of human serum the binding of the anti-AA 8, 7, 27 and 52to 5342-1204-4D11 gradually decreased (FIG. 4).

These data showed that a combination of these antibodies can be used todevelop an assay to measure the concentration of 5342-1204-4D11 in humanplasma. Such an assay would be useful in methods to measureconcentrations of 5342-1204-4D11, for example, in treated subjects.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

What is claimed is:
 1. An isolated antibody or fragment thereof thatbinds an activatable antibody or conjugated activatable antibody.
 2. Theisolated antibody of claim 1, wherein the antibody or fragment thereofhas an equilibrium dissociation constant of about 100 nM or less forbinding to the activatable antibody or conjugated activatable antibody.3. The isolated antibody of claim 1, wherein the antibody or fragmentthereof is a monoclonal antibody, domain antibody, single chain, Fabfragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, or a single domain light chain antibody.
 4. Theisolated antibody of claim 1, wherein the antibody or fragment thereofis a rabbit, mouse, chimeric, humanized or fully human monoclonalantibody.
 5. The isolated antibody of claim 1, wherein the antibody orfragment thereof comprises a combination of a variable heavy chaincomplementarity determining region 1 (CDRH1) sequence, a variable heavychain complementarity determining region 2 (CDRH2) sequence, a variableheavy chain complementarity determining region 3 (CDRH3) sequence, avariable light chain complementarity determining region 1 (CDRL1)sequence, a variable light chain complementarity determining region 2(CDRL2) sequence, and a variable light chain complementarity determiningregion 3 (CDRL3) sequence, wherein at least one CDR sequence is selectedfrom the group consisting of a VH CDR1 sequence that includes at leastan amino acid sequence selected from the group consisting of SEQ ID NO:67, 73, 78, 88, 95, and 101; a VH CD2 sequence that includes at least anamino acid sequence selected from the group consisting of SEQ ID NO: 68,74, 79, 89, 96, and 102; a VH CDR3 sequence that includes at least anamino acid sequence selected from the group consisting of SEQ ID NO: 69,80, 90, and 97; a VL CDR1 sequence that includes at least an amino acidsequence selected from the group consisting of SEQ ID NO: 70, 81, and98; a VL CDR2 sequence that includes at least an amino acid sequenceselected from the group consisting of SEQ ID NO: 71, 82, and 99; and aVL CDR3 sequence that includes at least an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 72, 83, 84, 91, 110, 100, and103.
 6. The isolated antibody of claim 1, wherein the antibody orfragment thereof comprises a combination of a VH CDR1 sequence, a VHCDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:67, 73, 78, 88, 95, and 101; the VH CD2 sequence comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 68, 74, 79,89, 96, and 102; the VH CDR3 sequence comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 69, 80, 90, and 97; theVL CDR1 sequence comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 70, 81, and 98; the VL CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 71, 82, and 99; and the VL CDR3 sequence comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 72, 83,84, 91, 110, 100, and
 103. 7. The isolated antibody of claim 1, whereinthe antibody comprises a variable heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO: 42, 56, 60, 64, 77, and106.
 8. The isolated antibody of claim 1, wherein the antibody comprisesa variable light chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 44, 58, 62, 66, 87, 94, and
 109. 9. Theisolated antibody of claim 1, wherein the antibody comprises a variableheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 42, 56, 60, 64, 77, and 106 and a variable light chain aminoacid sequence selected from the group consisting of SEQ ID NO: 44, 58,62, 66, 87, 94, and
 109. 10. The isolated antibody of claim 1, whereinthe antibody comprises a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and
 105. 11. Theisolated antibody of claim 1, wherein the antibody comprises a lightchain amino acid sequence selected from the group consisting of SEQ IDNO: 4, 8, 12, 16, 86, 93, and
 108. 12. The isolated antibody of claim 1,wherein the antibody comprises a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 6, 10, 14, 76, and105, and a light chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 4, 8, 12, 16, 86, 93, and
 108. 13. The isolatedantibody of claim 1, wherein the antibody comprises a combination of aheavy chain and a light chain selected from the group consisting of: (a)a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 and alight chain comprising the amino acid sequence of SEQ ID NO: 4; (b) aheavy chain comprising the amino acid sequence of SEQ ID NO: 6 and alight chain comprising the amino acid sequence of SEQ ID NO: 8; (c) aheavy chain comprising the amino acid sequence of SEQ ID NO: 10 and alight chain comprising the amino acid sequence of SEQ ID NO: 12; and (d)a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and alight chain comprising the amino acid sequence of SEQ ID NO:
 16. 14. Theisolated antibody of claim 1, wherein the antibody comprises acombination of a variable heavy chain and a variable light chainselected from the group consisting of: (a) a variable heavy chaincomprising the amino acid sequence of SEQ ID NO: 42 and a variable lightchain comprising the amino acid sequence of SEQ ID NO: 44; (b) avariable heavy chain comprising the amino acid sequence of SEQ ID NO: 56and a variable light chain comprising the amino acid sequence of SEQ IDNO: 58; (c) a variable heavy chain comprising the amino acid sequence ofSEQ ID NO: 60 and a variable light chain comprising the amino acidsequence of SEQ ID NO: 62; and (d) a variable heavy chain comprising theamino acid sequence of SEQ ID NO: 64 and a variable light chaincomprising the amino acid sequence of SEQ ID NO:
 66. 15. The isolatedantibody of claim 1, wherein the antibody comprises an antibody selectedfrom the group consisting of antibody 10, antibody 8, antibody 53,antibody 7, antibody 36, antibody 52, antibody, antibody 27, andantigen-binding fragments thereof.
 16. The isolated antibody of claim 1comprising an agent conjugated to the antibody or fragment thereof. 17.The isolated antibody of claim 16, wherein the agent is conjugated tothe antibody or fragment thereof via a linker.
 18. The isolated antibodyof claim 17, wherein the linker is a cleavable linker.
 19. The isolatedantibody of claim 17, wherein the linker is a non-cleavable linker. 20.The isolated antibody of claim 1 comprising a detectable moiety.
 21. Theisolated antibody of claim 20, wherein the detectable moiety is adiagnostic agent.
 22. An isolated nucleic acid molecule encoding theisolated antibody of claim
 1. 23. The isolated nucleic acid molecule ofclaim 22, wherein the nucleic acid encoding the isolated antibodycomprises a nucleic acid encoding a signal peptide.
 24. The isolatednucleic acid molecule of claim 23, wherein the nucleic acid encoding thesignal peptide is operably linked to the nucleic acid encoding theactivatable antibody via a nucleic acid encoding a spacer.
 25. A vectorcomprising the isolated nucleic acid molecule of claim
 22. 26. A methodof producing an isolated antibody by culturing a cell under conditionsthat lead to expression of the isolated antibody, wherein the cellcomprises the vector of claim 25.